Specific and high affinity binding proteins comprising modified SH3 domains of Fyn kinase

ABSTRACT

The present invention relates to a method for the production of a library comprising recombinant derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO: 1 as well as a method for selecting from a library comprising recombinant derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO: 1 one or more of said derivatives having a specific binding affinity to a protein or peptide.

FIELD OF THE INVENTION

The present invention relates to a method for the production of alibrary comprising recombinant derivatives of the SH3 domain of the Fynkinase of SEQ ID NO: 1 as well as a method for selecting from a librarycomprising recombinant derivatives of the SH3 domain of the Fyn kinaseof SEQ ID NO: 1 one or more of said derivatives having a specificbinding affinity to a protein or peptide.

The Sequence Listing submitted in text format (.txt) filed on Sep. 19,2014, named “S1236 US1 Sequenzprotokoll 4010961.txt”, (created on Sep.11, 2014, 364 KB), is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Specific and high-affinity binding agents are indispensable tools forbiological and medical research and also have utility for medicaldiagnosis, prophylaxis and treatment. At present, monoclonal antibodiesare the predominant class of binding molecules that can be rapidlyisolated with high affinity and specificity to virtually any target.However, immunoglobulins have limitations that are based mostly on theirgeneral biophysical properties and their rather complicated molecularstructure. Therefore, already in the 1990's several research groups haveexplored small globular proteins as substitutes for antibodies. The ideabehind this concept is the transfer of a universal binding site from anantibody structure to alternative protein frameworks, the so-calledscaffolds. So far more than 40 scaffolds have been described, among themtwo SH3 domains, the SH3 domains of the Abl and the Src kinase (see Binzet al., Nature Biotechnology, Vol. 23, No. 10, 1257-1268, 2005).

SH3 domains are found in many different proteins involved inintracellular signalling and cytoskeletal organization (Cohen et al.,“Modular binding domains in signal transduction proteins.” Cell 80(2):237-48, 1995). Despite the variability in their primary structures theseSH3 domains share a very similar overall structure and mode of bindingto proteins sharing the minimal consensus sequence PxxP that is acritical determinant for natural SH3 binding. An important function ofSH3 domains is to participate in highly selective protein-proteininteractions.

Erpel et al. (“Mutational analysis of the Src SH3 domain: the sameresidues of the ligand binding surface are important for intra- andintermolecular interactions.” Embo J. 14(5): 963-75, 1995) investigatedthe influence of mutations in the RT and n-Src loops of Src SH3 domainsand demonstrated that mutations in both loops which are adjacent to thehydrophobic surface could influence the ability of this domain toparticipate in inter- and intramolecular associations.

Hiipakka et al. (“SH3 domains with high affinity and engineered ligandspecificities targeted to HIV-1 Nef.” J. Mol. Biol. 293(5): 1097-106,1999) investigated the ability of the RT-loop of the Hck SH3 domain toact as a versatile specificity and affinity determinant. The authorsconstructed a phage library of Hck domains, where 6 amino acids of theRT-Loop were randomized (termed RRT-SH3). Using this strategy theyidentified individual RRT-SH3 domains that can bind to HIV-1 Nef up to40 times better than Hck-Sh3. The authors indicate the importance of theRT loop in SH3 ligand selection as a general strategy for creating SH3domains with desired binding properties.

Lee et al. (“A single amino acid in the SH3 domain of Hck determines itshigh affinity and specificity in binding to HIV-1 Nef protein.” Embo. J.14(20): 5006-15, 1995) investigated the structural basis of thedifferent SH3 binding affinities and specificities of Hck to the HIV-1Nef protein and were able to transfer the binding property of Hck SH3towards Nef to the Fyn SH3 domain by a single mutation in the RT loop ofthe Fyn SH3 domain (R96I).

Hosse et al. (“A new generation of protein display scaffolds formolecular recognition”, Protein Science, 15:14-27, 2006) specificallyaddress the requirements for binding proteins suitable for therapeuticapplications. The authors note the importance of some characteristicsfor therapeutically useful binding proteins such as serum stability,tissue penetration, blood clearance, target retention and immuneresponse. In the latter respect it is noted that non-human therapeuticproteins should be made as similar to their human counterparts aspossible and a human scaffold might be less immunogenic right from thestart. These authors conclude:

-   -   “However, even an entirely human scaffold is no guarantee for a        protein that does not elicit a human immune response, especially        if it is an intracellular protein. Randomization of amino acids        during library construction can potentially introduce novel        T-cell epitopes. Even single point mutations can render a human        protein immunogenic. Furthermore, most human scaffolds cause        some autoimmune response.”

Today, the SH3 domains of Abl and Hck kinases are acknowledged asprotein scaffolds for generating protein binders with prescribedspecificity, even though only binders towards known ligands like the Nefproteins or synthetic peptides have been identified so far (see Binz etal. above).

The SH3 domain of the Fyn kinase (Fyn SH3) comprises 63 residues (aa83-145 of the sequence reported by Semba et al. (“yes-relatedprotooncogene, syn, belongs to the protein-tyrosine kinase family.”Proc. Natl. Acad. Sci. USA 83(15): 5459-63, 1986) and Kawakami et al.(“Isolation and oncogenic potential of a novel human src-like gene.” MolCell Biol. 6(12): 4195-201, 1986). Fyn is a 59 kDa member of the Srcfamily of tyrosine kinases. As a result of alternative splicing the Fynprotein exists in two different isoforms differing in their kinasedomains; one form is found in thymocytes, splenocytes and somehematolymphoid cell lines, while a second form accumulates principallyin brain (Cooke and Perlmutter, “Expression of a novel form of the Fynproto-oncogene in hematopoietic cells.” New Biol. 1(1): 66-74, 1989).The biological functions of Fyn are diverse and include signalling viathe T cell receptor, regulation of brain function as well as adhesionmediated signalling (Resh, M. D. “Fyn, a Src family tyrosine kinase.”Int. J. Biochem. Cell Biol. 30(11): 1159-62, 1998). It is anintracellular protein. SEQ ID NO: 1 shows the Fyn SH3 sequence (aa83-145 of Fyn kinase as reported by Kawakami et al. and Semba et al. in1986, see above):

(SEQ ID NO: 1) GVTLFVALYDYEARTEDDLSFHKGEKFQILNSSEGDWWEARSLTTGETGYIPSNYVAPVDSIQ

The sequence of the RT-Src and the n-Src loop are underlined anddouble-underlined, respectively.

The amino acid sequence of Fyn SH3 is fully conserved among man, mouse,rat and monkey (gibbon). Chicken Fyn SH3 differs in one, the one ofXenopus laevis in two amino acid positions from the corresponding humandomain. Just as other SH3 domains the Fyn SH3 is composed of twoantiparallel β-sheets and contains two flexible loops (called RT-Src andn-Src-loops) in order to interact with other proteins.

In summary, the prior art teaches protein frameworks, the so-calledscaffolds, as alternatives to established antibody structures. The Srchomology 3 domain (SH3) is one of these about 40 or more scaffolds.Among the many different SH3 domains (about 300 in the human genome andseveral thousands described so far in nature) the Fyn SH3 is one, whichhas been used once before in order to elucidate SH3 binding specificityand affinity in general. The skilled person is also aware thatintracellular proteins are particularly prone to produce immuneresponses and, therefore, are typically less useful or even useless forin vivo applications like therapy and diagnosis.

The object underlying the present invention is to provide improvedtarget specific and high affinity binding proteins that are suitable asresearch, and in particular, as diagnostic and medical agents.Furthermore, these binding proteins should be stable and soluble underphysiological conditions, elicit little or no immune effects in humansreceiving these, and provide a binding structure that is also accessibleby large target structures, i.e. that is not masked by steric hindrance.

DESCRIPTION OF THE INVENTION

It was surprisingly found that the SH3 domain of the Fyn kinase of theSrc family provides excellent properties for designing recombinantbinding domains with specificity and high affinity to selected targets.In particular, it was found that the target specificity can be designedby mutating the RT loop and/or the src loop resulting in highervariability and improved binding properties for many targets.

Moreover, it was unexpectedly found that not only the native Fyn SH3binding protein but also mutated Fyn SH3-derived binding proteins werenot immunogenic in vivo. Therefore, recombinant mutant Fyn SH3 bindingproteins are particularly useful for the development of non-immunogenicprotein therapeutics and/or diagnostics.

As a result of the above, a first aspect the present invention relatesto a recombinant binding protein comprising at least one derivative ofthe Src homology 3 domain (SH3) of the Fyn kinase, wherein

-   -   (a) at least one amino acid in or positioned up to two amino        acids adjacent to the src loop and/or    -   (b) at least one amino acid in or positioned up to two amino        acids adjacent to the RT loop    -   is substituted, deleted or added, wherein the SH3 domain        derivative has an amino acid sequence having at least 70,        preferably at least 80, more preferably at least    -   90 and most preferred at least 95% sequence identity to the        amino acid sequence of SEQ ID NO: 1,    -   preferably with the proviso that the recombinant binding protein        does not comprise the amino acid sequence of SEQ ID NO: 2,    -   and preferably that the recombinant protein is not a natural SH3        domain containing protein existing in nature.

The amino acid sequence of SEQ ID NO: 2 (the Fyn SH3 variant R96I of Leeet al., see above) is provided below.

(SEQ ID NO: 2) GVTLFVALYDYEAITEDDLSFHKGEKFQILNSSEGDWWEARSLTTGETGYIPSNYVAPVDSIQ

In the context of this invention the RT loop of the Fyn kinase(sometimes also designated RT-Src-loop) consists of the amino acids E AR T E D that are located in positions 12 to 17 in SEQ ID NO: 1. Thepositions to be substituted, deleted and/or added, i.e. to be mutated,in or adjacent to the RT loop are amino acids 10 to 19, preferably 11 to18, more preferably 12 to 17.

In the context of this invention the src loop of the FYN kinase(sometimes also designated n-Src-loop) consists of the amino acids N S SE that are located in positions 31 to 34 in SEQ ID NO: 1. The positionsto be substituted, deleted and/or added, i.e. to be mutated, in oradjacent to the src loop are amino acids 29 to 36, preferably 30 to 35,more preferably 31 to 34.

The recombinant protein of the invention is preferably not a natural SH3domain containing protein existing in or isolated from nature. In otherwords, the scope of the invention preferably excludes wild type SH3domain containing proteins. There are abundant SH3 domain containingproteins in nature. These natural SH3 proteins have a binding affinityto their natural ligands. Most if not all of these natural SH3 ligandshave a PxxP motif. However, the recombinant proteins of the inventionare engineered proteins designed for having affinities to non-naturaltargets, i.e. non-natural targets being any target, e.g. in nature,preferably in a mammalian, more preferably in a human, excluding natural(wild-type) SH3 ligands. More preferably, the recombinant proteins ofthe invention essentially have no binding affinity to any natural SH3binding ligands, most preferably not to any natural SH3 binding ligandhaving a PxxP motif.

Preferably, the number of amino acids to be added into one and/or bothloops is 1 to 20, more preferably 1 to 10 or 1 to 5 amino acids, andmost preferably no amino acids are added into the loops.

In another preferred embodiment, the portions of the SH3 domainderivative that lie outside the RT and src loops are conserved as muchas possible in order not to introduce immunogenic motifs.

It is preferred that the recombinant proteins of the inventionessentially do not elicit an immunogenic reaction in mammals, preferablyin mouse, rat and/or human, most preferably in human. Of course, theimmunogenicity of the complete recombinant protein of the invention willnot only depend on the SH3 domain derivative portion but can beinfluenced by other portions of the whole protein.

In a preferred embodiment of the invention, at least the SH3 domainderivative portion of the recombinant protein is essentiallynon-immunogenic in mammals, preferably in mouse, rat and/or human, mostpreferably in human.

For example, the person skilled in the art can determine immunogenicreactions of the recombinant protein or its SH3 domain derivativeportion by standard and routine techniques, e.g. by administering (e.g.i.v. injection) a recombinant protein of interest or its SH3 domainderivative to a mammal such as a mouse and analysing the response ofimmunogenic blood cells and/or factors (e.g. interleukins) after anappropriate time for an immune reaction to occur.

In a more preferred embodiment the binding protein according to theinvention is one, wherein said SH3 domain derivative has at least 70 orat least 85, preferably at least 90, more preferably at least 95, mostpreferably at least 98 to 100% identity to the Src homology 3 domain(SH3) of the FYN kinase outside the src and RT loops.

In a preferred embodiment mutations are introduced in both the RT andsrc loops.

In a further more preferred embodiment the binding protein of theinvention comprises one or preferably two altered residues in positions37 and/or 50 of the SH3 domain derivative, preferably two hydrophobicaltered residues, more preferably Trp37 and/or Tyr50, Trp37 and Tyr50being most preferred. As demonstrated in FIG. 3b below theirrandomization can increase the affinity.

The term “derivative of the Src homology 3 domain (SH3) of the FYNkinase”, as it is used herein, is meant to encompass an amino acidsequence having at least 70, preferably at least 80, more preferably atleast 90 and most preferred at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 1. The same meaning holds true for an SH3domain derivative having at least 70 or at least 85, preferably at least90, more preferably at least 95, most preferably at least 98% identityto the Src homology 3 domain (SH3) of the FYN kinase outside the src andRT loops, except that the amino acids forming said loops are excludedwhen determining the sequence identity.

For the purpose of determining the extent of sequence identity of aderivative of the Fyn SH3 domain to the amino acid sequence of SEQ IDNO: 1, for example, the SIM Local similarity program can be employed(Xiaoquin Huang and Webb Miller, “A Time-Efficient, Linear-Space LocalSimilarity Algorithm.” Advances in Applied Mathematics, vol. 12:337-357, 1991.), freely available from the authors and their institute(see also the world wide web:http://www.expasy.org/tools/sim-prot.html); for multiple alignmentanalysis ClustalW can be used (Thompson et al., “CLUSTAL W: improvingthe sensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weight matrixchoice.”, Nucleic Acids Res., 22(22): 4673-4680, 1994.). Preferably, theextent of the sequence identity of the derivative to SEQ ID NO: 1 isdetermined relative to the complete sequence of SEQ ID NO: 1.

In a preferred embodiment the binding protein of the invention comprisesat least two derivatives of the Fyn SH3 domain. More preferably, it is abivalent binding protein. The at least two derivatives of the SH3 domainmay be the same or different. Preferably, they are the same.

The binding protein of the invention can be designed to have anyspecific binding affinity to a given target. In a preferred embodiment,the target is an amino acid-based target such as a peptide or protein,more preferably one comprising a PxxP motif. Of course, only a minorityof natural and physiologically relevant target proteins contains a PxxPmotif. The examples below demonstrate that binding proteins according tothe invention for targets (e.g. ED-B domain of fibronectin, interleukin(IL) 17A, the serine protease chymase (E.C. 3.4.21.39), human epidermalgrowth factor receptor 2 (Her-2), human serum albumin, the transmembranereceptor CD33, epidermal growth factor receptor (EGFR) and themembrane-bound aspartic protease BACE (UniProt Q95YZ0)) with motifsother than PxxP are available. Therefore, the binding protein of theinvention is by no means limited to the PxxP motif and can have aspecific binding affinity to any given target, e.g. sugars,polypeptides, etc.

More preferably, the binding protein according to the invention has aspecific binding affinity to a target of 10⁻⁷ to 10⁻¹² M, preferably10⁻⁸ to 10⁻¹² M, preferably a therapeutically and/or diagnosticallyrelevant target, more preferably an amino acid-based target comprising aPxxP motif.

In a most preferred aspect, the binding protein according to theinvention has a specific (in vivo and/or in vitro) binding affinity of10⁻⁷ to 10⁻¹² M, preferably 10⁻⁸ to 10⁻¹² M, to the extracellular domainof oncofetal fibronectin (ED-B).

In a preferred embodiment, the present invention relates to arecombinant binding protein, comprising at least one derivative of theSrc homology 3 domain (SH3) of the FYN kinase, wherein

-   -   (a) at least one amino acid in or positioned up to two amino        acids adjacent to the src loop and/or    -   (b) at least one amino acid in or positioned up to two amino        acids adjacent to the RT loop,        is substituted, deleted or added,        wherein the SH3 domain derivative has an amino acid sequence        having at least 70, preferably at least 80, more preferably at        least 90 and most preferred at least 95% sequence identity to        the amino acid sequence of SEQ ID NO: 1,        preferably with the proviso that the recombinant binding protein        does not comprise the amino acid sequence of SEQ ID NO: 2,        and preferably with the proviso that the recombinant protein is        not a natural SH3 domain containing protein existing in nature,        wherein said binding protein has a specific (in vivo and/or in        vitro) binding affinity of preferably 10⁻⁷ to 10⁻¹² M, more        preferably 10⁻⁸ to 10⁻¹² M, to the extracellular domain of        oncofetal fibronectin (ED-B).

In a more preferred embodiment said SH3 domain derivative has at least85, preferably at least 90, more preferably at least 95, most preferablyat least 98 to 100% identity to the Src homology 3 domain (SH3) of theFYN kinase outside the src and RT loops.

In another more preferred embodiment the above ED-B-specific bindingprotein comprises at least two derivatives of the SH3 domain, preferablyit is a bivalent binding protein.

Preferably, said ED-B-specific binding protein has one or more,preferably two, altered, preferably hydrophobic, residues in positions37 and/or 50 of the SH3 domain derivative, in particular Trp37 and/orTyr50, Trp37 and Tyr50 being most preferred.

Next to a specific binding affinity to polypeptide and protein targets,the binding protein of the invention can also have a specific bindingaffinity to a small organic or non-amino-acid based compound, e.g. asugar, oligo- or polysaccharide, fatty acid, etc.

A number of antibody-cytokine fusion proteins have already beeninvestigated for applications in, e.g. arthritis or cancer therapy,often with impressive results. For example, the human antibody L19specific to the ED-B domain of fibronectin (a marker of angiogenesis)has been used to deliver pro-inflammatory cytokines (such as IL-2, IL-12or TNF) to solid tumours, sometimes with striking therapeutic benefits[for a review and corresponding references see Neri & Bicknell, Nat.Rev. Cancer (2005) 5: 436-446, and also WO 01/62298].

The binding protein of the present invention now allows for substitutingantibodies in prior art fusion proteins and also for designing new andless immunogenic fusion proteins for in vivo and in vitro pharmaceuticaland diagnostic applications.

In a second aspect, the invention relates to a fusion protein comprisinga binding protein of the invention fused to a pharmaceutically and/ordiagnostically active component.

A fusion protein of the invention may comprise non-polypeptidecomponents, e.g. non-peptidic linkers, non-peptidic ligands, e.g. fortherapeutically or diagnostically relevant radionuclides.

Preferably, said active component is a cytokine, preferably a cytokineselected from the group consisting of IL-2, IL-12, TNF-alpha, IFN alpha,IFN beta, IFN gamma, IL-10, IL-15, IL-24, GM-CSF, IL-3, IL-4, IL-5,IL-6, IL-7, IL-9, IL-11, IL-13, LIF, CD80, B70, TNF beta, LT-beta, CD-40ligand, Fas-ligand, TGF-beta, IL-1 alpha and IL-1 beta.

More preferably, said active component is a toxic compound, preferably asmall organic compound or a polypeptide, preferably a toxic compoundselected from the group consisting of calicheamicin, neocarzinostatin,esperamicin, dynemicin, kedarcidin, maduropeptin, doxorubicin,daunorubicin, auristatin, Ricin-A chain, modeccin, truncated Pseudomonasexotoxin A, diphtheria toxin and recombinant gelonin.

In another preferred embodiment, the fusion protein according toinvention is one, wherein said active component is a chemokine,preferably a chemokine selected from the group consisting of IL-8, GROalpha, GRO beta, GRO gamma, ENA-78, LDGF-PBP, GCP-2, PF4, Mig, IP-10,SDF-1alpha/beta, BUNZO/STRC33, I-TAC, BLC/BCA-1, MIP-1alpha, MIP-1 beta,MDC, TECK, TARC, RANTES, HCC-1, HCC-4, DC-CK1, MIP-3 alpha, MIP-3 beta,MCP-1-5, Eotaxin, Eotaxin-2, I-309, MPIF-1, 6Ckine, CTACK, MEC,Lymphotactin and Fractalkine.

In a further preferred embodiment the binding protein according to theinvention contains artificial amino acids.

In further preferred embodiments of the fusion protein of the presentinvention said active component is a fluorescent dye, preferably acomponent selected from the groups of Alexa Fluor or Cy dyes (Berlier etal., “Quantitative Comparison of Long-wavelength Alexa Fluor Dyes to CyDyes: Fluorescence of the Dyes and Their Bioconjugates”, J HistochemCytochem. 51 (12): 1699-1712, 2003.); a photosensitizer, preferablybis(triethanolamine)Sn(IV) chlorin e₆ (SnChe₆); a pro-coagulant factor,preferably tissue factor; an enzyme for pro-drug activation, preferablyan enzyme selected from the group consisting of carboxy-peptidases,glucuronidases and glucosidases; a radionuclide either from the group ofgamma-emitting isotopes, preferably ^(99m)Tc, ¹²³I, ¹¹¹In, or from thegroup of positron emitters, preferably ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ¹²⁴I, orfrom the group of beta-emitter, preferably ¹³¹I, ⁹⁰Y, ¹⁷⁷Lu, ⁶⁷Cu, orfrom the group of alpha-emitter, preferably ²¹³Bi, ²¹¹At; and/or afunctional Fc domain, preferably a human functional Fc domain.

The above mentioned functional Fc domain will allow for directing amammal's immune response to a site of specific target binding of thebinding protein component of the fusion protein, e.g. in therapeutic,prophylactic and/or diagnostic applications.

A further preferred embodiment relates to fusion proteins according tothe invention as mentioned above, further comprising a componentmodulating serum half-life, preferably a component selected from thegroup consisting of polyethylene glycol (PEG), immunoglobulin andalbumin-binding peptides.

In a most preferred embodiment, the fusion protein of the invention asmentioned above comprises a binding protein of the invention having aspecific (in vivo and/or in vitro) binding affinity of 10⁻⁷ to 10⁻¹² M,preferably 10⁻⁸ to 10⁻¹² M, to the extra domain of oncofetal fibronectin(ED-B). Preferably, said ED-B-specific binding protein has one or more,preferably two hydrophobic residues in positions 37 and/or 50 of the SH3domain derivative, in particular Trp37 and/or Tyr50, Trp37 and Tyr50being most preferred.

Binding and fusion proteins according to the invention may be preparedby any of the many conventional and well known techniques such as plainorganic synthetic strategies, solid phase-assisted synthesis techniquesor by commercially available automated synthesizers. On the other hand,they may also be prepared by conventional recombinant techniques aloneor in combination with conventional synthetic techniques.

Further aspects of the present invention are directed to (i) apolynucleotide coding for a binding protein or fusion protein accordingto the invention, (ii) a vector comprising said polynucleotide, (iii) ahost cell comprising said polynucleotide and/or said vector.

Polynucleotides can be DNA, RNA, PNA and any other analogues thereof.The vectors and host cells may be any conventional type that fits thepurpose, e.g. production of binding and fusion proteins of theinvention, therapeutically useful vectors and host cells, e.g. for genetherapy. The skilled person will be able to select thosepolynucleotides, vectors and host cells from an abundant prior art andconfirm their particular suitability for the desired purpose by routinemethods and without undue burden.

The binding and fusion proteins of the present invention do not elicit astrong and preferably have essentially no immune response in mammals, inparticular in humans and mice, as was demonstrated for mice and isanalogously expected to hold true for humans, too, because the Fyn SH3is identical in both mammalian species. It was surprisingly found thatneither native Fyn SH3 nor mutated Fyn SH3 causes an immune response inmice injected i.v. with either one. This was unexpected because Fynkinase is an intracellular protein and does not participate in neonatalB cell selection. Therefore, Fyn SH3-derived binding and fusion proteinswith designed target specificity and affinity are particularly wellsuited for therapeutic, prophylactic and/or diagnostic applications invivo.

Hence, a highly relevant aspect of the present invention relates to theuse of a binding or fusion protein according to the invention forpreparing a medicament.

In a further aspect, the binding or fusion protein of the invention isused for preparing a diagnostic means, in particular for in vivoapplications.

Preferably, an ED-B specific binding or fusion protein as describedabove is used for preparing a medicament or diagnostic means for thetreatment or diagnosis of cancer.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising a binding or fusion protein of the invention andoptionally a pharmaceutically acceptable excipient.

Another aspect of the present invention relates to a diagnosticcomposition, preferably for in vivo applications, comprising a bindingor fusion protein of the invention and optionally a pharmaceuticallyacceptable excipient.

Preferably, the pharmaceutical or diagnostic composition comprises anED-B specific binding or fusion protein of the invention and optionallya pharmaceutically acceptable excipient.

Pharmaceutical compositions and diagnostic means for in vivoapplications of the present invention typically comprise atherapeutically or diagnostically effective amount of a binding and/orfusion protein according to the invention and optionally auxiliarysubstances such as pharmaceutically acceptable excipient(s). Saidpharmaceutical compositions are prepared in a manner well known in thepharmaceutical art. A carrier or excipient may be a liquid materialwhich can serve as a vehicle or medium for the active ingredient.Suitable carriers or excipients are well known in the art and include,for example, stabilizers, antioxidants, pH-regulating substances,controlled-release excipients. The pharmaceutical preparation of theinvention may be adapted, for example, for parenteral use and may beadministered to the patient in the form of solutions or the like.

Finally, another aspect of the present invention concerns a method oftreatment or diagnosis, wherein an effective amount of the abovepharmaceutical or diagnostic composition is administered to a patient inneed thereof, preferably a patient suffering or suspected of sufferingfrom cancer and/or inflammatory diseases.

In effecting treatment or diagnosis of a subject suffering fromdiseases, a binding or fusion protein of the present invention can beadministered in any form or mode which makes the therapeutic ordiagnostic compound bioavailable in an effective amount, including oralor parenteral routes. For example, compositions of the present inventioncan be administered subcutaneously, intramuscularly, intravenously andthe like. One skilled in the art in the field of preparing formulationscan readily select the proper form and mode of administration dependingupon the particular characteristics of the product selected, the diseaseor condition to be treated or diagnosed, the stage of the disease orcondition and other relevant circumstances (see. e.g. Remington'sPharmaceutical Sciences, Mack Publishing Co. (1990)). The compositionsof the present invention can be administered alone or in the form of apharmaceutical or diagnostic preparation in combination withpharmaceutically acceptable carriers or excipients, the proportion andnature of which are determined by the solubility and chemical propertiesof the product selected, the chosen route of administration and standardpharmaceutical and diagnostic practice. The products of the presentinvention, while effective themselves, may be formulated andadministered in the form of their pharmaceutically acceptable salts,such as acid addition salts or base addition salts, for purposes ofstability, convenience of crystallization, increased solubility and thelike.

FIGURES

FIG. 1A illustrates a dot blot analysis for FynSH3 mutants withrandomized RT-Src-loop. The percentage of clones expressing a detectableamount of soluble Fyn SH3 mutants was determined by dot blot analysis ofbacterial cell lysates using anti-HIS-HRP antibody conjugate (Sigma) asdetecting reagent. Peroxidase activity was detected using the ECL plusWestern blotting detection system (Amersham).

FIG. 1B illustrates a dot blot analysis for FynSH3 mutants with anextended (4→6) and randomized n-Src-loop. The percentage of clonesexpressing a detectable amount of soluble Fyn SH3 mutants was determinedby dot blot analysis of bacterial cell lysates using anti-HIS-HRPantibody conjugate (Sigma) as detecting reagent. Peroxidase activity wasdetected using the ECL plus Western blotting detection system(Amersham).

FIG. 1C illustrates a dot blot analysis for FynSH3 with RT- and n-Srcrandomized loops. The percentage of clones expressing a detectableamount of soluble Fyn SH3 mutants was determined by dot blot analysis ofbacterial cell lysates using anti-HIS-HRP antibody conjugate (Sigma) asdetecting reagent. Peroxidase activity was detected using the ECL plusWestern blotting detection system (Amersham).

FIG. 2 illustrates a monoclonal phage-ELISA. After the third round ofpanning against MSA monoclonal bacterial supernatants containing phagesdisplaying Fyn SH3 mutants were tested by ELISA using MaxiSorp plates(Nunc) coated with MSA (100 μg/ml overnight, 100 μl per well). Boundphages were detected using anti M-13-HRP antibody conjugates (Amersham).

FIG. 3A illustrates monoclonal phage-ELISA (against MSA) after one roundof affinity maturation selection using MaxiSorp plates (Nunc) coatedwith MSA (100 μg/ml overnight, 100 μl per well) for Phage ELISA of thefirst sub-library of G4 (randomized n-Src loop and Trp37 and Tyr50). Theparental clone G4 is indicated with an arrow.

FIG. 3B illustrates monoclonal phage-ELISA (against MSA) after one roundof affinity maturation selection using MaxiSorp plates (Nunc) coatedwith MSA (100 μg/ml overnight, 100 μl per well) for Phage ELISA of thesecond sub-library of G4 (randomized and extended n-Src loop). Theparental clone G4 is indicated with an arrow.

FIG. 3C illustrates monoclonal phage-ELISA (against MSA) after one roundof affinity maturation selection using MaxiSorp plates (Nunc) coatedwith MSA (100 μg/ml overnight, 100 μl per well) for Phage ELISA of thefirst and second sub-library after one round of panning, performed underconditions favouring binders with a long k_(off). The parental clone G4is indicated with an arrow.

FIG. 4 shows the soluble ELISA (using MaxiSorp plates (Nunc) coated withMSA (100 μg/ml overnight, 100 μl per well) of several MSA bindingclones, after cloning (pQE-12 vector), expression and purification ofthe soluble protein, according to the manufacturer's instructions(Qiagen, native conditions). As detecting agents anti-HIS-HRP antibodyconjugates were used. As a control the same binding proteins were addedto wells blocked with 4% MPBS only.

FIG. 5 Specificity ELISA of soluble protein. Selected MSA binding FynSH3 mutants were tested for binding against human serum albumin (HSA),rat serum albumin (RSA), bovine serum albumin (BSA) and ovalbumin usingMaxiSorp plates (Nunc) coated with the different albumins (each 100μg/ml overnight, 100 μl per well).

FIG. 6 BIACore analysis of D3. Used concentrations: 4, 2, 1, and 0.5 μM(from top).

FIG. 7A ELISA analysis of blood samples for the presence of murineantibodies. MaxiSorp plates (Nunc) were coated with Fyn SH3 (20 μg/mlovernight, 100 μl per well). Blood samples (ranging from 75-200 μl) ofeach of the 5 mice were applied in dilution series (from 1:4 to 1:100).Detection of antibodies was performed using anti-mouse-IgG-HRP antibodyconjugate (Sigma). As a control of the coating efficiencyanti-HIS-HRP-conjugates (Sigma) were used.

FIG. 7B ELISA analysis of blood samples for the presence of murineantibodies. MaxiSorp plates (Nunc) were coated with Fyn SH3 D3 (20 μg/mlovernight, 100 μl per well). Blood samples (ranging from 75-200 μl) ofeach of the 5 mice were applied in dilution series (from 1:4 to 1:100).Detection of antibodies was performed using anti-mouse IgG-HRP antibodyconjugate (Sigma). As a control of the coating efficiencyanti-HIS-HRP-conjugates (Sigma) were used.

FIG. 7C ELISA analysis of blood samples for the presence of murineantibodies. MaxiSorp plates (Nunc) were coated with scFv (60 μg/mlovernight, 100 μl per well). Blood samples (ranging from 75-200 μl) ofeach of the 4 mice were applied in dilution series (from 1:4 to 1:100).Detection of antibodies was performed using anti-mouse-IgG-HRP antibodyconjugate (Sigma). As a control of the coating efficiency,anti-myc-HRP-conjugates (Roche) were used.

FIG. 8A shows immunofluorescence of D3 on F9 murine teratocarcinomahistological sections.

FIG. 8B shows the corresponding negative control of FIG. 8A on F9 murineteratocarcinoma histological sections.

FIG. 8C shows the anti-CD31 staining on F9 murine teratocarcinomahistological sections.

FIG. 8D shows the corresponding negative control of FIG. 8C on F9 murineteratocarcinoma histological sections.

FIG. 9A shows the tumor retention of Fyn SH3-D3. Targeting results areexpressed as % injected dose of ¹²⁵I-labeled protein retained per g oftissue (% ID/g).

FIG. 9B shows that no accumulation could be observed for Fyn SH3 wt.Targeting results are expressed as % injected dose of ¹²⁵I-labeledprotein retained per g of tissue (% ID/g).

FIG. 10. shows the SDS PAGE analysis of embodiments of IL-17-bindingpolypeptides of the invention: (a) SDS PAGE of B1_2 (SEQ ID No: 42)(lane 1), E4 (SEQ ID NO: 60) (lane 2), 2C1 (SEQ ID NO: 110) (lane 3),E4-Fc (SEQ ID NO: 120) (lane 4: non-reducing conditions, lane 5:reducing conditions), 2C1-Fc (SEQ ID NO: 121) (lane 6: non-reducingconditions, lane 7: reducing conditions); (b) SDS PAGE of [(2C1)2-Fc](SEQ ID NO: 122) (lane 1: non-reducing conditions, lane 2: reducingconditions). The molecular weight of (2C1)2-Fc is estimated from thereference molecular weight full range marker (not shown).

FIG. 11 shows size exclusion chromatograms (SEC) of IL-17A-bindingpolypeptides of the invention: (a) Clone B1_2 (SEQ ID NO: 42), (b) E4(SEQ ID NO: 60), (c) 2C1 (SEQ ID NO: 110), (d) E4-Fc (SEQ ID NO: 120),(e) SEC-peak purified E4-Fc, analyzed after 40 days after purificationand storage in PBS at 4° C., (f) 2C1-Fc (SEQ ID NO: 121), (g) (2C1)2-Fc(SEQ ID NO: 122).

FIG. 12 depicts BIAcore sensograms of IL-17A-binding polypeptides of theinvention: (a) Clone B1_2 (SEQ ID NO: 42), (b) E4 (SEQ ID NO: 60), (c)2C1 (SEQ ID NO: 110), (d) E4-Fc (SEQ ID NO: 120), (e) 2C1-Fc (SEQ ID NO:121), f) (2C1)₂-Fc (SEQ ID NO: 122).

FIG. 13 depicts the results of an IL-17A inhibition cell assay: (a)Dose-dependent induction of IL-6 after incubation of NHDF cells withIL-17A. (b) Dose-dependent inhibition of IL-17A-induced IL-6 productionin NHDF cells by Fyn SH3 derived IL-17 binders and IL-17A receptor-Fcchimera. (c) same as b), Fyn SH3 wt protein was used as a controlprotein with no IL-17A binding affinity. (d) XTT-assay: viable cells areable to metabolize the tetrazolium salt XTT to a coloured product. Inour experiment, all cells were viable after 24 hours incubation withIL-17A, IL-17A and Fyn SH3 binders, or IL-17A and IL-17R-Fc chimera.

FIG. 14 depicts a size exclusion chromatography with an IL-17A-bindingpolypeptide of the invention designated G3 (SEQ ID NO: 37) one day afterpurification (stored in PBS at 4° C.). The chromatography was performedusing a Superdex 75 (GE Healthcare) column.

FIG. 15A depicts a size exclusion chromatography with an IL-17A-bindingpolypeptide of the invention designated G3 (SEQ ID NO: 37) stored formore than six months at 4° C.

FIG. 15B depicts a size exclusion chromatography with an IL-17A-bindingpolypeptide of the invention designated G3 (SEQ ID NO: 37) stored formore than six months at −20° C.

FIG. 16A shows the pharmacokinetic data of an IL-17A-binding polypeptideof the invention designated E4-Fc (SEQ ID NO: 120) in mice, where E4-Fcconcentration in serum is plotted versus time after intravenousinjection. The last four time points were used to calculate the terminalhalf-life of 50.6 hours.

FIG. 16B shows the pharmacokinetic data of an IL-17A-binding polypeptideof the invention designated E4-Fc (SEQ ID NO: 120) in mice, where E4-Fcconcentration in serum is plotted versus time after intravenousinjection, but with a semi-logarithmic display. The last four timepoints were used to calculate the terminal half-life of 50.6 hours.

FIG. 17 shows a table of the binding specificity of a polypeptide of theinvention designated 2C1 (SEQ ID NO: 110). The absorbance results relateto an ELISA performed using different target proteins: human IL-17A,human IL-17F, mIL-17A (murine IL-17 A), TNF-alpha (human tumor necrosisfactor alpha), BSA (bovine serum albumin), Ovalbumin (hen egg white),IL-6 (human interleukin 6).

FIG. 18 shows the specificity of the Fyn SH3-derived polypeptide of theinvention 2C1 (SEQ ID NO: 110). Different IL-17 family members, IL-17Aof different species and other unrelated antigens were used in ELISAwith the Fyn SH3-derived polypeptide of the invention 2C1 (SEQ ID NO:110) as binding agent. Fyn SH3-derived polypeptide of the invention 2C1(SEQ ID NO: 110) only binds to human and cynomolgus IL-17A. No bindingto any of the other antigens could be detected. On the right side of theFigure (right side of the dashed line) the ELISA signal to human IL-17Cis shown, which was determined on another day with the human IL-17Acontrol. Legend: hIL-17A: human Interleukin 17A, hIL-17B: humanInterleukin 17B, hIL-17D: human Interleukin 17D, hIL-17E: humanInterleukin 17E, hIL-17F: human Interleukin 17F, mouse IL-17A: mouseInterleukin 17A, rat IL-17A: rat Interleukin 17A, canine IL-17A: canineInterleukin 17A, cyno II-17A: cynomolgus Interleukin 17A, EDB: extradomain B of fibronectin, hIL-6: human Interleukin 6, hTNF alpha: humanTumor Necrosis Factor alpha, Ovalbumin: Albumin from chicken egg white,BSA: Bovine Serum Albumin neg ctrl: no antigen was used for coating,hIL-17C: human Interleukin 17C.

FIG. 19 depicts the Biacore sensogram of the Fyn SH3-derived polypeptideof the invention 2C1 (SEQ ID NO: 110) on a chip coated with cynomolgusIL-17A refolded from inclusion bodies.

FIG. 20 shows SDS PAGE analysis of Fc fusion proteins. Lane 1: fullrange rainbow marker (GE Healthcare), lane 2: 2C1-Fc (SEQ ID NO: 133),lane 3: full range rainbow marker (GE Healthcare), lane 4:2C1-m5-Fc(LALA) (SEQ ID NO: 136), lane 5: 2C1-m10-Fc(LALA) (SEQ ID NO:137), lane 6: 2C1-m15-Fc(LALA) (SEQ ID NO: 138), lane 7:2C1-m5E-Fc(LALA) (SEQ ID NO: 135), lane 8: 2C1-Fc(LALA) (SEQ ID NO:134).

FIG. 21 shows the ELISA of 2C1-Fc (SEQ ID NO: 133) binding to IL-17Aafter storage for 5 days at 37° C. in human serum (▪) compared to thestandard control 2C1-Fc (SEQ ID NO: 133) stored at 4° C. in PBS (x).

FIG. 22 shows the serum concentration at different time-points of2C1-Fc(LALA) (SEQ ID NO: 134) after a single i.v. injection into mice.2C1-Fc(LALA) fusion protein (SEQ ID NO: 134) produced in mammalian cellswas injected (40 μg per animal) intravenously (iv) (n=5) in mice. Thelast four time points of the PK profile were used to calculate aterminal half-life of 2C1-Fc fusion protein of 53 hours.

FIG. 23 shows the inhibition of human IL-17A induced KC production bythe anti-IL-17 Fyn SH3-derived polypeptide 2C1 (SEQ ID NO: 110) of theinvention in an acute inflammation model. Two hours after s.c. injectionof either 3 μg human IL-17A (IL-17), PBS (PBS), 3 μg human IL-17A with17 μg monomeric Fyn SH3-derived polypeptide 2C1 (SEQ ID NO: 110) of theinvention (IL-17+2C1), 3 μg human IL-17A with 16 μg wild-type Fyn SH3monomer (IL-17+wt), 17 μg monomeric Fyn SH3-derived polypeptide 2C1 (SEQID NO: 110) of the invention alone (2C1), or 16 μg wild-type Fyn SH3monomer alone (wt), blood samples were taken and KC levels inmouse-serum were quantified. Mean KC levels of 4 mice per group areshown (±SD), with the exception of the wild-type control groups (Fyn SH3without and with IL-17A), where mean levels of 3 mice are shown (±SD).

FIG. 24 depicts the inhibition of human IL-17A induced KC production by2C1-Fc fusion protein (SEQ ID NO: 133) in an acute inflammation model.2C1-Fc/IL-17: 44 μg of 2C1-Fc (SEQ ID NO: 133) was injected i.v.followed by s.c. injection of 3 μg human IL-17A. Two hours afteradministration of IL-17A, blood samples were taken from the mice and KCserum levels were measured by ELISA. Control experiments were performedas follows: PBS/IL-17: i.v. injection of PBS followed by s.c. injectionof IL-17; 2C1-Fc/PBS: i.v. injection of 2C1-Fc (SEQ ID NO: 133) followedby s.c. injection of PBS; PBS/PBS: i.v. injection of PBS followed bys.c. injection of PBS; Mean KC levels of 3-5 mice per group are shown(±SD).

FIG. 25 shows the ELISA signals for binding of the indicated FynSH3-derived polypeptides of the invention to chymase. No ELISA signalscould be detected for the binding to the irrelevant protein bovine serumalbumin (BSA).

FIG. 26 shows the monomeric size exclusions profiles of the followingFyn SH3-derived polypeptides of the invention: A) Fyn SH3-derivedpolypeptide of the invention F12 (SEQ ID NO: 5), B) Fyn SH3-derivedpolypeptide of the invention G2.3 (SEQ ID NO: 157), C) Fyn SH3-derivedpolypeptide of the invention E3 (SEQ ID NO: 160), D) Fyn SH3-derivedpolypeptide of the invention B5 (SEQ ID NO: 154), E) Fyn SH3-derivedpolypeptide of the invention D7 (SEQ ID NO: 158), E) Fyn SH3-derivedpolypeptide of the invention E4 (SEQ ID NO: 153), F) Fyn SH3-derivedpolypeptide of the invention H2 (SEQ ID NO: 159), G) Fyn SH3-derivedpolypeptide of the invention A4 (SEQ ID NO: 155)

FIG. 27 FACS binding experiments using HER2 overexpressing BT-474 cells.

-   -   (A) Binding of Fyn SH3 derived polypeptides C12 (SEQ ID NO: 167)        and G10 (SEQ ID NO: 168) on HER2 with or without pre-blocking of        the epitope of the anti-HER2 antibody 1 (anti-HER2 mAb 1;        wherein the heavy chain has the amino acid sequence of SEQ ID        NO: 320 and the light chain has the amino acid sequence of SEQ        ID NO: 321; exemplary nucleic acid molecules encoding the heavy        and light chain are shown in SEQ ID NO: 331 and 332) and        anti-HER2 antibody 2 (anti-HER2 mAb 2; wherein the heavy chain        has the amino acid sequence of SEQ ID NO: 326 and the light        chain has the amino acid sequence of SEQ ID NO: 329; exemplary        nucleic acid molecules encoding the heavy and light chain are        shown in SEQ ID NO: 334 and 335). PBS, phosphate buffered        saline, represents the negative control.    -   (B) Binding of biotinylated anti-HER2 antibody 1 and        biotinylated anti-HER2 antibody 2 (biotinylated antibodies are        indicated with the abbreviation “bt”) with or without        pre-blocking of the epitope of the anti-HER2 antibody 1 and        anti-HER2 antibody 2. PBS, phosphate buffered saline, represents        the negative control.

FIG. 28 In vitro proliferation assays with HER2 overexpressing gastriccancer cell line NCl-N87.

-   -   Fyn SH3-derived polypeptide C12 (SEQ ID NO: 167) was fused to        the Fc part of a human IgG1 to create the monospecific bivalent        protein called Fc-C12 (SEQ ID NO: 319). The combination mixture        of Fynomer C12-Fc with the anti-HER2 antibody 1 (anti-HER2        mAb 1) (shown in FIG. 2A) and with the anti-HER2 antibody 2        (anti-HER2 mAb 2) (shown in FIG. 2C) did not reduce        proliferation rate of NCl-N87 cells more effectively than the        corresponding anti-HER2 antibodies alone. However, the        anti-proliferative activity of the binding molecules COVA208        (SEQ ID NO: 319 & 325) (shown in FIG. 2B) and COVA210 (SEQ ID        NO: 326 & 327; an exemplary nucleic acid molecule encoding SEQ        ID NO: 327 is shown in SEQ ID NO: 336) (FIG. 2D) was higher than        the activity of the corresponding unmodified antibody. COVA 208        consists of the fusion of C12 (SEQ ID NO: 167) to the N-terminus        of the light chain of antibody 1 (SEQ ID NO: 320 and 321) and        COVA210 consists of the fusion of C12 (SEQ ID NO: 167) to the        N-terminus of the light chain of antibody 2 (SEQ ID NO: 226 and        329), see also FIG. 8.

FIG. 29 The anti-proliferative activity of anti-HER2 Fynomer-antibodyfusions varies depending on the relative orientation of the Fynomer andthe binding site of the antibody.

-   -   The anti-proliferative activities of the different        Fynomer-antibody fusion proteins in a proliferation cell assay        with NCl-N87 gastric cancer cells showed variations (A) and (B),        and COVA208 showed the best anti-proliferative effects on this        cell line (FIG. 3B). The maximal effects are indicated in the        tables and given in percentage of viability. COVA201 (SEQ ID        NOs: 322 and 321), COVA202 (SEQ ID NOs: 320 and 323), COVA207        (SEQ ID NOs: 324 and 321) and COVA208 (SEQ ID NOs: 320 and 325)        are all fusion proteins of the Fyn SH3 derived polypeptide C12        (SEQ ID NO: 167) and anti-HER2 antibody 1 (anti-HER2 mAb 1) (SEQ        ID NOs: 320 and 321). COVA201 consists of the C-terminal heavy        chain fusion, COV202 represents the C-terminal light chain        fusion, COVA 207 consists of the N-terminal heavy chain fusion        and COVA208 represents the N-terminal light chain fusion, see        also FIG. 8.

FIG. 30 The anti-proliferative activity of COVA208 (SEQ ID NOs: 321 and325) (fusion of Fynomer C12 to the N-terminus of the light chain ofanti-HER2 antibody 1 (anti-HER2 mAb 1, SEQ ID NOs: 320 and 321)) wasdetermined in a cell assay with the HER2 overexpressing breast cancercell line BT-474. COVA208 exhibited superior anti-proliferative activityas compared to the unmodified antibody.

FIG. 31 depicts an animal study with a NCl-N87 gastric cancer xenograftmouse model. NCl-N87 gastric cancer cells were inoculated subcutaneouslyin CD1 Nude mice (n=6 per treatment group). When tumors reached a sizeof about 140 mm³, animals were treated with a loading dose of 30 mg/kgCOVA208 (SEQ ID NOs: 320 and 325), anti-HER2 antibody 1 (anti-HER2 mAb 1(SEQ ID NOs: 320 and 321)) or placebo (PBS). Treatment was continuedwith four weekly i.p. injections (15 mg/kg) (indicated with the arrows)and size of tumors was measured with a caliper. COVA208 was found toinhibit tumor growth significantly better than the monospecificanti-HER2 antibody 1 or placebo (PBS). Mean tumor volumes of 6 mice areshown (relative to day 0 when the treatment was started)±standard errorof the mean (SEM).

FIG. 32 Serum concentrations of COVA208 (SEQ ID NOs: 320 and 325) andthe anti-HER2 antibody 1 (anti-HER2 mAb 1 (SEQ ID NOs: 320 and 321)) atdifferent time-points after a single i.v. injection into C57Bl/6 mice.The six last time-points were used to calculate the terminal half-livesof 247 h (COVA208) and 187 h (anti-HER2 antibody 1). Mean serumconcentrations are plotted versus time, error bars represent standarddeviations (SD).

FIG. 33 SDS PAGE of COVA208 (SEQ ID NOs: 320 and 325) and anti-HER2antibody 1 (anti-HER2 mAb 1 (SEQ ID NO: 320 and 321)) (top) and sizeexclusion chromatograms of COVA208 after purification and after astorage period of 1 and 2 months at 4° C. (bottom). Evidently, COVA208did not form any aggregates.

FIG. 34 In vitro proliferation assays with HER2 expressing cell lines.COVA208 (SEQ ID NOs: 320 and 325) inhibited the cell growth of OE19(FIG. 9A) and of Calu-3 cells (FIG. 9B) more effectively than anti-HER2antibody 1 (anti-HER2 mAb 1 (SEQ ID NOs: 320 and 321)). FIG. 9Csummarizes the results of the in vitro proliferation assays performed on10 different cell lines, for each of which the maximal level ofinhibition has been plotted. The corresponding data points for COVA208and anti-HER2 antibody 1 were connected to facilitate the comparisonbetween the two compounds.

-   -   COVA208 shows improved inhibition of cell growth as compared to        anti-HER2 antibody 1 on all 10 cell lines.

FIG. 35 COVA208 (SEQ ID NOs: 320 and 325) is capable of inducingapoptosis, as determined by caspase-3/7 activity (FIG. 10A) and by TUNELstaining (FIG. 10B). Anti-HER2 antibody 1 (anti-HER2 mAb 1 (SEQ ID NOs:320 and 325)) did not increase caspase-3/7 activity nor the fraction ofTUNEL-positive cells, indicating that the ability to induce apoptosis isunique to COVA208. Staurosporine was used as positive control. Errorbars in FIG. 10A indicate standard deviation of triplicates.

FIG. 36 COVA208 (SEQ ID NOs: 320 and 325) inhibits ligand-dependentactivation of HER2 signaling on MCF-7 cells (left panel) as well asligand-independent activation of HER2 signaling on NCl-N87 cells (rightpanel). Anti-HER2 antibody 1 (anti-HER2 mAb 1 (SEQ ID NOs: 320 and 321))inhibits signaling only on MCF-7 cells, whereas anti-HER2 antibody 2(anti-HER2 mAb 2 (SEQ ID NOs: 226 and 329)) is only active on NCl-N87cells. Vinculin served as a loading control.

FIG. 37 COVA208 is internalized by NCl-N87 cells. After surface stainingfollowed by 5 h incubation, 52% of COVA208 (SEQ ID NOs: 320 and 325) wasfound in spherical dots within the cytosol, as determined from confocallaser scanning images analyzed with Imaris software. Anti-HER2 antibody1 (anti-HER2 mAb 1 (SEQ ID NOs: 320 and 321)) staining primarilyremained membrane-associated, with only 9% of the staining localized incytosolic spherical dots.

FIG. 38 depicts an animal study with a KPL-4 breast cancer xenograftmouse model. KPL-4 breast cancer cells were inoculated subcutaneously inSCID beige mice (n=8 per treatment group). When tumors reached a size ofabout 70 mm³, animals were treated with a loading dose of 30 mg/kgCOVA208 (SEQ ID NOs: 320 and 325), anti-HER2 antibody 1 (anti-HER2 mAb 1(SEQ ID NOs: 320 and 321)) or placebo (PBS). Treatment was continuedwith four weekly i.p. injections (15 mg/kg) (indicated with the arrows)and size of tumors was measured with a caliper. COVA208 was found toinhibit tumor growth significantly better than the monospecificanti-HER2 antibody 1 or placebo (PBS). Mean tumor volumes of 8 mice areshown ±standard error of the mean (SEM).

FIG. 39 shows the SDS-PAGE characterization of albumin-bindingpolypeptides of the invention: Lane M: molecular weight standard; LaneA: Fynomer® C1 (SEQ ID NO: 440); Lane B: Fynomer® 17H (SEQ ID NO: 441);Lane C: WT Fyn-SH3 (SEQ ID NO: 339).

FIG. 40 shows the SDS-PAGE characterization of the unmodified BITE®polypeptide (SEQ ID NO: 378, lane 1) and the Fynomer®-BITE® fusionprotein COVA406 (SEQ ID NO: 379, lane 2), consisting of the albuminbinding Fynomer® 17H and the BITE® molecule. The molecular weightstandard is shown in Lane M.

FIG. 41 shows the size exclusion chromatogram (SEC) of COVA406 (SEQ IDNO: 379).

FIG. 42 depicts a FACS binding experiment with COVA406 (SEQ ID NO: 379)using cells expressing CD3 (Jurkat E6-1), cells expressing PSMA (22Rv1cells) and an irrelevant cell line expressing neither CD3 nor PSMA(LS174T cells). Binding is expressed as mean fluorescence intensity.CD3+: CD3-positive cells; CD3-: CD3-negative cells; PSMA+: PSMA-positivecells; PSMA-: PSMA-negative cells; COVA406: COVA406 is used as bindingreagent; PBS: negative control, phosphate buffered saline is addedinstead of COVA406. COVA406 recognizes both antigens CD3 and PSMAexpressed on cells.

FIG. 43 depicts the analysis of redirected cell lysis of PSMA positivecells (22Rv1 cells) or PSMA negative cells (HT29 cells) by COVA406 (SEQID NO: 379) using human PBMCs as effector cells. The target cells 22Rv1and HT29 were pre-labeled with Calcein AM and then incubated with humanPBMCs (at effector cell to target cell ratio (E:T) of 25:1) anddifferent concentrations of COVA406 (SEQ ID NO: 379) for 5 hours. Thepercentage of specific tumor cell lysis was measured by detection ofcalcein-release into the supernatant. Triplicates of 3 wells are shown±SEM.

FIG. 44 shows the serum concentrations of COVA406 (SEQ ID NO: 379) andthe BITE® protein (SEQ ID NO: 378) at different time-points after asingle i.v. injection into C57BL/6 mice. The concentration in serum wasdetermined by ELISA. Mean values of 5 mice are shown ±SD

FIG. 45 Specificity ELISA of prior art Fyn SH3 variants isolated afteraffinity selections. MSA=mouse serum albumin, HSA=human serum albumin,RSA=rat serum albumin, BSA=bovine serum albumin

FIG. 46 shows the SDS-PAGE characterization of Fynomer®-antibody fusionproteins of the invention and control proteins. A: gel run undernon-reducing conditions; B: gel run under reducing conditions. Lane M:molecular weight standard; Lane 1: anti-CD3 antibody (SEQ ID NOs 381 and382); Lane 2: COVA420 (SEQ ID NOs 387 and 388); Lane 3: COVA446 (SEQ IDNO: 391).

FIG. 47 shows the SDS-PAGE characterization of an Fynomer®-antibodyfusion proteins of the invention. A: gel run under non-reducingconditions; B: gel run under reducing conditions. Lane M: molecularweight standard; Lane 1: COVA422 (SEQ ID NOs: 389 and 390)

FIG. 48 shows size exclusion (SEC) profiles of Fynomer®-antibody fusionproteins. A: SEC profile of COVA420 (SEQ ID NOs 387 and 388) on FPLCsystem; B: SEC profile of COVA422 (SEQ ID NOs: 389 and 390) on HPLCsystem

FIG. 49A shows the flow cytometric binding analysis of Fynomer®-antibodyfusion proteins COVA420 (SEQ ID NOs: 387 and 388); COVA422 (SEQ ID NOs389 and 390), and the bispecific scFv-control COVA446 (SEQ ID NO: 391)on HER2 positive cells. Signals are compared to the background signalobtained with the secondary detection antibody only (grey shadedhistograms).

FIG. 49B shows the flow cytometric binding analysis of Fynomer®-antibodyfusion proteins COVA420 (SEQ ID NOs: 387 and 388); COVA422 (SEQ ID NOs389 and 390), and the bispecific scFv-control COVA446 (SEQ ID NO: 391)on CD3 positive cells. Signals are compared to the background signalobtained with the secondary detection antibody only (grey shadedhistograms).

FIG. 49C shows the flow cytometric binding analysis of Fynomer®-antibodyfusion proteins COVA420 (SEQ ID NOs: 387 and 388); COVA422 (SEQ ID NOs389 and 390), and the bispecific scFv-control COVA446 (SEQ ID NO: 391)on cells that do not express HER2 nor CD3. Signals are compared to thebackground signal obtained with the secondary detection antibody only(grey shaded histograms).

FIG. 50 shows the redirected cell kill activity of COVA420 (SEQ ID NOs:387 and 388) and the bispecific anti-CD3×anti-HER2 control in singlechain Fv format (COVA446, SEQ ID NO: 391) on HER2 positive SKBR-3 tumorcells. In addition, the absence of any kill activity of COVA420 on HER2negative MDA-MB-468 cells is shown, demonstrating the specific killactivity towards HER2 positive cells. PBMCs were used as effector cells.

FIG. 51 shows the redirected cell kill activity of COVA420 (SEQ ID NOs:387 and 388) and COVA422 (SEQ ID NOs 389 and 390) on HER2 positiveSKOV-3 tumor cells using CD8+ enriched T-cells as effector cells.

FIG. 52 depicts the release of Granzyme B into the cell culturesupernatant upon incubation of indicated antibody Fynomer® fusionproteins in the presence and absence of CD8+ enriched T-cells.

FIG. 53 shows the serum concentrations of COVA420 (SEQ ID NOs: 387 and388), after intravenous injection in mice.

FIG. 54 shows the anti-tumor activity of COVA420 (SEQ ID NOs: 387 and388) in an in vivo SKOV-3 xenograft model reconstituted with activatedand expanded human T-cells. Tumor volumes are presented as RTV (relativetumor volume to day of therapy start). 0.5 mg/kg COVA420 and vehicletreatments were administered twice weekly (day 6, 9, 13, 15), andequimolar doses of COVA446 (SEQ ID NO: 391) (0.16 mg/kg) by dailyintravenous (i.v.) bolus injections. Black arrows visualize doseintervals.

FIG. 55 shows the redirected cell kill activity of COVA420 (SEQ ID NOs387 and 388) and the bispecific anti-CD3×anti-HER2 control in singlechain Fv format (COVA446, SEQ ID NO: 391) towards SKOV-3 tumor cellsexpressing high level of HER2 and MCF-7 tumor cells expressing low levelof HER2. CD8+ enriched T-cells were used as effector cells. The percentof remaining target cell viability is shown.

FIG. 56 shows size exclusion (SEC) profiles of Fynomer-antibody fusionproteins. A: SEC profile of COVA493 (SEQ ID NOs 393 and 395); B: SECprofile of COVA494 (SEQ ID NOs: 393 and 421); C: SEC profile of COVA497(SEQ ID NOs: 422 and 395); D: SEC profile of COVA499 (SEQ ID NOs: 394and 423); E: SEC profile of COVA489 (SEQ ID NOs: 394 and 395).

FIG. 57 shows the flow cytometric binding analysis of the anti-CD3antibody (COVA489, SEQ ID NOs: 394 and 395), the Fynomer®-antibodyfusion proteins COVA493 (SEQ ID NOs 393 and 395), COVA494 (SEQ ID NOs:394 and 421), COVA497 (SEQ ID NOs: 422 and 395), COVA499 (SEQ ID NOs:394 and 423) and the bispecific anti-CD3×anti-EGFR control in singlechain Fv format (COVA445, SEQ ID NO: 396) on EGFR positive cells(MDA-MB-468, upper panel) and on CD3 positive cells (Jurkat E6-1. lowerpanel). Signals are compared to the background signal obtained with thesecondary detection antibody only (grey shaded histograms).

FIG. 58 shows the redirected cell kill activity of COVA493 (SEQ ID NOs393 and 394), COVA494 (SEQ ID NOs: 394 and 421), COVA497 (SEQ ID NOs:422 and 395), COVA499 (SEQ ID NOs: 394 and 423) and the bispecificanti-CD3×anti-EGFR control in single chain Fv format (COVA445, SEQ IDNO: 396) towards MDA-MB-468 tumor cells expressing high level of EGFRand HT-29 tumor cells expressing low level of EGFR. In addition, theabsence of any kill activity of the anti-CD3 antibody (COVA489, SEQ IDNOs: 394 and 395) at the highest concentration of 5 nM is shown. CD8-Eenriched T-cells were used as effector cells. The percent of remainingtarget cell viability is shown.

FIG. 59 shows the redirected cell kill activity of COVA493 (SEQ ID NOs393 and 394), COVA494 (SEQ ID NOs: 394 and 421), COVA497 (SEQ ID NOs:422 and 395), COVA499 (SEQ ID NOs: 394 and 423) and the bispecificanti-CD3×anti-EGFR control in single chain Fv format (COVA445, SEQ IDNO: 396) towards MDA-MB-468 and HT-29 tumor cells, expressing high andlow level of EGFR respectively, in the presence or absence of effectorT-cells. (n.d).: not determined.

FIG. 60 (A) shows size exclusion (SEC) profiles of the Fynomer-antibodyfusion protein COVA467 (SEQ ID NOs: 381 and 398) (B) shows theredirected cell kill activity of COVA467 (SEQ ID NOs: 381 and 398) andthe bispecific anti-CD3×anti-CD33 control in single chain Fv format(COVA463, SEQ ID NO: 399) on CD33 positive U937 tumor cells. Inaddition, the absence of any kill activity of the unmodified anti-CD3antibody (COVA419, SEQ ID NOs: 381 and 382) is shown. CD8+ enrichedT-cells were used as effector cells. The percent of target cell lysis isshown.

In the following the subject-matter of the invention will be describedin more detail referring to specific embodiments which are not intendedto be construed as limiting to the scope of the invention.

EXAMPLES Example 1 Anti-ED-B Fyn SH3 Derivative Example 1.1 Expressionof Fyn SH3 Mutants

For the purpose of evaluating the expression of mutants of Fyn SH3 a dotblot analysis of three different Fyn SH3 sublibraries was performed(FIG. 1): in the first library only the RT-loop was randomized, in thesecond the Src loop was randomized and extended to 6 residues and in thethird library the RT- and the Src loop were randomized simultaneously,the latter loop being extended from 4 to 6 residues. The percentage ofexpressed Fyn SH3 mutants ranged from 59-90%.

TABLE 1 Library Expressed mutants (%) Number of clones tested RT-Src 5929 n-Src 90 29 RT-Src and n-Src 62 58

Example 1.2 Phage Display Selections Against Mouse Serum Albumin

A library of 10⁷ different Fyn SH3 was created (only the RT-loop wasrandomized) and cloned into the phagemid vector pHEN1 (Hoogenboom et al.“Multi-subunit proteins on the surface of filamentous phage:methodologies for displaying antibody (Fab) heavy and light chains”,Nucleic Acids Res, 19(15):4133-7, 1991). The library was displayed onphages and 3 rounds of panning were performed against mouse serumalbumin (MSA).

After the third round, screening for binding proteins was performed bymonoclonal phage-ELISA; 13 positive clones were detected (FIG. 2).Sequencing of the 13 clones revealed that two different sequences wereenriched, denoted G4 and C4.

However, after subcloning and expression of G4 in the pQE-12 vector(Qiagen, expression and purification according to manufacturer'shandbook under native conditions) the binding of the protein towards MSAcould not be detected by ELISA (FIG. 4) due to low affinity (phage ELISAis more sensitive than the ELISA of the soluble protein). Therefore, thesequence of G4 was used for two different affinity maturation libraries(size: 10⁷ clones for each library). In the first one, the 4 residues ofthe n-Src loop and residues Trp37 (SEQ ID NO: 1) and Tyr50 (SEQ IDNO: 1) were randomized, in the second one the n-Src loop was extendedfrom 4 to 6 randomized residues. After one round of panning severalclones of both sublibraries gave stronger signals in Phage ELISAcompared to the parental clone G4 (FIG. 3). After subcloning andexpression of several clones the binding of the soluble protein wasconfirmed by ELISA (FIG. 4). Apparent dissociation constants were in therange of 100 nM (determined by BIAcore). Some of the clones werecross-reactive with other serum albumins (tested: human serum albumin(HSA), rat serum albumin (RSA), bovine serum albumin (BSA) andovalbumin), whereas other clones were highly specific to MSA, indicatingthat it is possible to isolate high specific binding proteins (FIG. 5).

Example 1.3 Phage Display Selections Against the Extra Domain b ofFibronectin (ED-B)

ED-B was chosen as a target protein in order to demonstrate the abilityto select Fyn SH3 derived binders against a pharmaceutically relevantprotein. ED-B is a 91 amino acid Type III homology domain that isinserted into the fibronectin molecule by a mechanism of alternativesplicing at the level of the primary transcript whenever tissueremodelling takes place (Zardi et al., “Transformed human cells producea new fibronectin isoform by preferential alternative splicing of apreviously unobserved exon.” Embo J. 6(8): 2337-42, 1987). It is a goodquality marker of angiogenesis that is overexpressed in a variety ofsolid tumors (e.g. renal cell carcinoma, colorectal carcinoma,hepatocellular carcinoma, high-grade astrocytomas, head and necktumours, bladder cancer, etc.) but is virtually undetectable in normaladult tissue (except for the endometrium in the proliferative phase andsome vessels in the ovaries). (For more details on ED-B as a target seeMenrad and Menssen, “ED-B fibronectin as a target for antibody-basedcancer treatments.” Expert Opin. Ther. Targets 9(3): 491-500, 2005).

A library of more than 1 billion Fyn SH3 mutants was prepared anddisplayed on phages (simultaneous randomization of RT-Src and n-Srcloops). After three rounds of panning against ED-B 3 binding clones wereidentified by phage ELISA. Sequencing revealed two different sequences(clones denoted B11 and D3). The dissociation constant of D3 wasdetermined by surface plasmon resonance real-time interaction analysisusing a BIAcore3000 instrument and showed a value of 8.5×10⁻⁸ M (FIG.6).

D3 (SEQ ID NO: 3) GVTLFVALYDYHAQSGADLSFHKGEKFQILKFGRGKGDWWEARSLTTGETGYIPSNYVAPVDSIQ

Example 1.4 Immunogenicity

Immunogenicity of proteins is one of the major drawbacks inprotein-related therapies, especially for treatments involvingrepetitive administrations of a drug. Due to the conservation of the FynSH3 sequence in mice and men the immunogenic potential of the FynSH3wild type protein (Fyn SH3 wt) and a Fyn SH3 mutant (Fyn SH3D3, a binderagainst ED-B) was investigated in vivo by injecting 5 mice repeatedlywith the two proteins. Mice were injected 4 times (every third day) with20 μg of protein. One day after the 4^(th) injection mice weresacrificed and blood samples were taken for examining the presence orabsence of murine anti-Fyn SH3 wt and anti-Fyn SH3D3 antibodies. As apositive control 4 mice were injected (equal time points of injectionand equal dosages (=60 μg)) with a human antibody in the single chain Fvformat (scFv). However, one mouse of the scFv group died 20 minutesafter the third injection and the other 3 were about to die, so bloodsamples were already taken after the third injection. FIGS. 7 a and bdemonstrate that there were no detectable antibodies against Fyn SH3 wtand Fyn SH3D3, whereas strong signals were observed for the controlgroup (FIG. 7c ).

Example 1.5 Immunohistofluorescence

In order to explore whether Fyn SH3-D3 (D3, a binder against ED-B)recognizes its target in the native conformation in the tissue,immunofluorescence on F9 teratocarcinoma sections was performed. FIG. 8illustrates that D3 bound the tumor stroma around blood vessels (FIG.8.a). The detection was performed with anti-His-Alexa488 antibodyconjugate. In the negative control, no D3 protein was added (FIG. 8b ).In order to visualize blood vessels, the same sections were co-stainedwith a rat anti-mouse-CD31 antibody and as a secondary antibody donkeyanti-rat Alexa594 conjugate was used (FIG. 8.c). The negative controlwas done using the secondary antibody without the primary antibody (FIG.8.d).

Example 1.6 Quantitative Biodistribution In Vivo

The in vivo targeting performance of Fyn SH3-D3 (a binder against ED-B)and Fyn SH3 wild type (a non-binder to ED-B) was evaluated bybiodistribution experiments in mice bearing a s.c. grafted F9 murineteratocarcinoma. Since ED-B is identical in mouse and man the results ofthe tumor targeting studies should be predictive of the D3 performancein humans. ¹²⁵I-labeled D3 and SH3 wt were injected i.v. and 24 h later,animals were sacrificed, the organs excised, weighed and radioactivitywas counted. FIG. 9.a shows that D3 selectively accumulated in the tumor(tumor:organ ratios ranged from 3:1 to 10:1), whereas no enrichmentcould be observed for the Fyn SH3 wild type protein (FIG. 9.b).

Example 2 Anti-IL17A Fyn SH3 Derivatives Example 2.1 Fyn SH3-DerivedPolypeptides of the Invention Bind to IL-17a as Determined by MonoclonalPhage ELISA

Methods

DNA encoding the amino acid sequences shown in SEQ ID NOs: 4 to 119 werecloned into the phagemid vector pHEN1 as described for the FYN SH3library in Grabulovski et al. (Grabulovski et al. (2007) JBC, 282, p.3196-3204). Phage production was performed according to standardprotocols (Viti, F. et al. (2000) Methods Enzymol. 326, 480-505).Monoclonal bacterial supernatants containing phages were used for ELISA:biotinylated IL-17A (purchased from R&D Systems, biotinylation wasperformed with NHS-PEO4-biotin (Pierce) according to the manufacturer'sinstructtions) was immobilized on streptavidin-coated wells(StreptaWells, High Bind, Roche), and after blocking with PBS, 2% milk(Rapilait, Migros, Switzerland), 20 μl of PBS, 10% milk and 80 μl ofphage supernatants were applied. After incubating for 1 h and washing,bound phages were detected with anti-M13-HRP antibody conjugate (GEHealthcare). The detection of peroxidase activity was done by adding BMblue POD substrate (Roche) and the reaction was stopped by adding 1 MH₂SO₄. The DNA sequence of the binders was verified by DNA sequencing(BigDye Terminator v3.1 cycle sequencing kit, ABI PRISM 3130 GeneticAnalyzer, Applied Biosystems).

Results

The amino acid sequences of Fyn SH3-derived IL-17A binders is presentedin SEQ ID NOs: 4 to 119 as appended in the sequence listing. All SEQ IDNOs bound IL-17A in this phage ELISA experiment.

Example 2.2 Fyn SH3-Derived Polypeptides of the Invention Bind toRecombinant Human IL-17 S with High Affinities

This example shows the cloning and expression of different formats ofFyn SH3-derived IL-17A-binding polypeptides, as well as thecharacterization of these polypeptides by size exclusion chromatographyand surface plasmon resonance experiments.

a) Cloning and Expression of Fyn SH3-Derived IL-17A-Binding Polypeptides

Selected Fyn SH3-derived IL-17A-binding polypeptides (clone B1_2: SEQ IDNO: 42, clone E4: SEQ ID NO: 60 and clone 2C1: SEQ ID NO: 110) werecloned into the cytosolic expression vector pQE-12 and expressed as wellas purified as described in Grabulovski et al. (Grabulovski et al.(2007) JBC, 282, p. 3196-3204).

b) Cloning and Expression of Fyn SH3-Derived IL-17A-Binding PolypeptidesFused to the Fc Part of a Human IgG1 Antibody

Clones E4 and 2C1 (SEQ ID NO: 60 and SEQ ID NO: 110) were cloned andexpressed as fusion proteins with the Fc part of a human IgG1 antibody(see below for procedure; SEQ ID NO: 120 and 121). Furthermore, a 2C1dimer with a 10 amino acid linker [(2C1)₂-Fc] was cloned and expressedas Fc fusion protein (SEQ ID NO: 122).

The Fc part of human IgG1 was PCR-amplified using the primers fm5 (5′ATCGGGA-TCCGACAAAACTCACACATGCC 3′, SEQ ID NO: 124) and fm6 (5′TACGAAGCTTT-CATTTACCCGGAGACAGGG 3′, SEQ ID NO: 125) and using thecommercial pFUSE-hIgG1-Fc2 (Invivogen) eukaryotic vector as template.The resulting PCR product was digested with BamHI/HindIII and ligatedwith the pASK-IBA2 vector (IBA-Biotagnology) previously digested withthe same enzymes, yielding the new vector pAF.

The genetic information of clones E4 and 2C1 (SEQ ID NO: 60 and SEQ IDNO: 110) was PCR amplified with fm7 (5′ATATCACCATGGGGCCGGAGTGACACTCTTTGTG-GCCCTTTATG 3′, SEQ ID NO: 126) andfm8 (5′ CGTAGGA-TCCCTGGATAGAGTC-AACTGGAGC 3′, SEQ ID NO: 127). For thepreparation of the 2C1 dimer fused to Fc, the 2C1 DNA template was usedfor two independent PCRs. In the first reaction the primers 47b.fo (5′AGA GCC ACC TCC GCC TGA ACC GCC TCC ACC CTG GAT AGA GTC AAC TGG AGC CAC3′, SEQ ID NO: 128) and 52. ba (5′ gac taa cga gat cgc gga tcc gga gtgaca ctc ttt gtg gcc ctt tat 3′, SEQ ID NO: 129) were used and in thesecond PCR primers 48b.ba (5′ GGT GGA GGC GGT TCA GGC GGA GGT GGC TCTGGA GTG ACA CTC TTT GTG GCC CTT TAT 3′, SEQ ID NO: 130) and 51. fo (5′ATC CCA AGC TTA GTG ATG GTG ATG GTG ATG CAG ATC CTC TTC TGA GAT GAG TTTTTG TTC ACC CTG GAT AGA GTC AAC TGG AGC CAC 3′, SEQ ID NO: 131) wereused.

The two DNA fragments were assembled by PCR, yielding a 2C1 homodimerwith a 10 amino acid linker (GGGGSGGGGS, SEQ ID NO: 123) between the twodomains. The resulting DNA fragment was further amplified as describedfor the 2C1 monomer using the primers fm7 and fm8. Obtained PCR productswere then digested with NcoI/BamHI and cloned into the double-digestedperiplasmic expression vector pAF. Plasmids were electroporated into E.coli TG1 and protein expression was induced with 0.2 μg/mlanhydrotetracyclin. Bacterial cultures were grown overnight at 25° C. ina rotary shaker and Fynomer-Fc fusion proteins were purified from theperiplasmic fraction in a single protein A-affinity chromatography step.SDS PAGE (Invitrogen) analysis was performed with 20 μl of proteinsolution.

c) Size Exclusion Chromatography (SEC)

Size Exclusion Chromatography (SEC) was performed on an ÄKTA FPLC systemusing a Superdex 75 column (10/300) or Superdex 75 Short Column (5/150)(GE Healthcare).

d) Affinity Measurements

Affinity measurements were performed using a BIAcore 3000 instrument(Biacore). For the interaction analysis between biotinylated IL-17A andmonomeric Fyn SH3-derived IL-17A-binding polypeptides, and betweenbiotinylated IL-17A and E4-Fc (SEQ ID NO: 120), a streptavidin SA chip(Biacore) was used with 1300 and 510 RU biotinylated IL-17A immobilized,respectively. The running buffer was PBS, 0.1% NaN₃ and surfactant P20(Biacore). The interactions were measured at a flow of 20 μl/min andinjection of different concentrations of Fyn SH3-derived IL-17A-bindingpolypeptides. For the interaction analysis between IL-17A and the 2C1-Fcfusions as well as the (2C1)₂-Fc fusion, a CM5 chip (Biacore) was coatedwith 2900 RU goat anti-human IgG Fc-specific antibody (JacksonImmunoresearch). The running buffer was HBS-EP (Biacore). Theinteractions were measured by injecting about 250 to 275 RU Fc fusionprotein at a flow rate of 10 μl/min, followed by injection of differentconcentrations of IL-17A (R&D Systems) at a flow rate of 30 μl/min. Allkinetic data of the interaction (separate kon/koff) were evaluated usingBIA evaluation 3.2RC1 software.

e) Results

The expression yields for monomeric Fyn SH3-derived IL-17A-bindingpolypeptides of the invention ranged from 60 to 85 mg/liter of bacterialculture under non-optimized conditions in shake flasks. The Fc-fusionproteins were expressed with a yield of 0.2 to 0.4 mg/liter (Table 2).The Fc-fusion proteins have the sequences listed in SEQ ID NOs: 120 to122 as appended)

TABLE 2 Expression yields after purification of bacterial culture undernon-optimized conditions in shake flasks in E. coli. Clone SEQ ID NO:Expression yield (mg/L) B1_2 42 65 E4 60 85 2C1 110 60 E4-Fc 120 0.42C1-Fc 121 0.3 [(2C1)₂-Fc] 122 0.2

FIG. 10 shows the SDS PAGE analysis of the indicated purified proteins.

Size exclusion chromatography (SEC) profiles demonstrated that allconstructs eluted mainly as single, monomeric peaks (see FIG. 11). Asalready observed in earlier studies for Fyn SH3-derived binding proteins(Grabulovski et al. (2007) JBC, 282, p. 3196-3204), the main peak eluteslater than expected for a protein of about 8 kDa. For the Fc-fusionproteins of the invention a second purification step by size exclusionchromatography was performed after the single-step protein A-sepharosepurification yielding monomeric proteins as shown for the fusion proteinE4-Fc (SEQ ID NO: 120) in FIG. 11e . E4-Fc (SEQ ID NO: 120) was stablefor at least 40 days when stored at 4° C. in PBS.

The binding properties were analyzed by real-time interaction analysison a BIAcore chip (FIG. 12) revealing the following dissociationconstants (K_(D)) for selected IL-17A-binding polypeptides and fusionproteins:

TABLE 2 Clone SEQ ID NO: K_(D) B1_2 42 117 nM E4 60 31 nM 2C1 110 5 nME4-Fc 120 5 nM 2C1-Fc 121 305 pM [(2C1)₂-Fc] 122 180 pM

Example 2.3 IL-17a Inhibition Cell Assay

IL-17A induces the production of IL-6 in fibroblasts in a dose-dependentmanner (Yao et al. (1995) Immunity, 3, p. 811-821). The inhibitoryactivities of the indicated Fyn SH3-derived IL-17A-binding polypeptidesand fusion proteins were tested by stimulating human dermal fibroblastswith recombinant IL-17A in the absence or presence of variousconcentrations of Fyn SH3 mutants or human IL-17A receptor-Fc chimera.Cell culture supernatants were taken after 24 h of stimulation andassayed for IL-6 with ELISA. In addition, a colorimetric test wasperformed using the reagent XTT in order to demonstrate that the cellswere viable after 24 h of incubation with IL-17A alone, or IL-17A andFyn SH3-derived inhibitory IL-17A-binding polypeptides of the invention,or IL-17A and IL-17R-Fc chimera. Only viable and metabolic active cellsare capable of reducing the tetrazolium salt XTT to orange-coloredcompounds of formazan (Scudiero, et al. (1988), Cancer Res. 48, p.4827-4833).

Methods

For endotoxin removal the indicated protein solutions were filteredthree times with the Acrodisc Mustang E membrane (VWR). After filtrationthe endotoxin levels of the protein solutions containing inhibitory FynSH3-derived IL-17A-binding polypeptides of the invention were less than0.1 EU/ml, as determined by the Limulus amebocyte lysate (LAL) test(PYROGENT Single test Gel Clot LAL Assay (Lonza)).

400 μl of a cell suspension containing about 1×10⁴ Normal Human DermalFibroblasts (PromoCell, NHDF-c, C12300) were distributed per well (24well plate, Nunc or TPP) and cultured for 24 hours at 37° C. (medium:Fibroblast Growth Medium C-23010, PromoCell). The supernatant wasaspirated and after mixing different concentrations of Fyn SH3 derivedIL-17A-binding polypeptides of the invention or IL-17A receptor Fcchimera (RnD Systems) with IL-17A (RnD Systems) containing medium (50ng/ml final concentration), 350 μl of the corresponding solution wasadded per well (mixing ratio between inhibitor solution andIL-17A-containing medium was 1:3). As a positive control PBS was mixedwith the IL-17A containing medium (“no inhibitor”) in a ratio of 1:3 andas a negative control PBS was mixed with medium only (“no IL-17A”) in aratio of 1:3. For the determination of the IL-17A-dependent IL-6production, IL-17A containing medium was used (final concentrations ofIL-17A: 10, 25 and 50 ng/ml) and mixed with PBS in a ratio of 3:1. After24 hours incubation at 37° C. the supernatant was aspirated and the IL-6concentration was determined by ELISA according to the manufacturer'sinstructions (IL-6 ELISA kit, R&D Systems). Immediately after theaspiration of the supernatant the XTT-containing medium was added (CellProliferation Kit II, Roche) and cell viability was determined accordingto the manufacturer's instructions.

The percentage of IL-17A inhibition was determined with the followingformula:

${{Inhibition}\mspace{14mu}(\%)} = {100 - \frac{\begin{pmatrix}{{A\; 450} - {650\mspace{14mu}{{nm}({sample})}} -} \\{{A\; 450} - {650\mspace{14mu}{{nm}( {{neg}.\mspace{14mu}{control}} )} \times 100}}\end{pmatrix}}{\begin{pmatrix}{{A\; 450} - {650\mspace{14mu}{{nm}( {{pos}.\mspace{14mu}{control}} )}} -} \\{{A\; 450} - {650\mspace{14mu}{{nm}( {{neg}.\mspace{14mu}{control}} )}}}\end{pmatrix}}}$Results

Normal Human Dermal Fibroblasts (NHDF) were incubated with IL-17A atdifferent concentrations. FIG. 13 (a) shows the IL-17A dose-dependentinduction of IL-6. In a next step NHDF cells were incubated with IL-17A(50 ng/ml) and different concentrations of indicated Fyn SH3-derivedIL-17A-binding polypeptides of the invention or IL-17A receptor-Fcchimera (FIG. 13(b)). It was observed that both clones 2C1 (SEQ ID NOD:110) and E4 (SEQ ID NO: 60) inhibited the IL-17A induced IL-6 productionwith IC₅₀ values of about 1 nM and 6 nM, respectively. The IL-17Areceptor-Fc chimera has a reported IC₅₀ value of 500 pM (R&D Systems).In this experiment, a value of about 1 nM was obtained. The assaydepicts a representative result of three independent experiments. Inorder to further demonstrate that the inhibition of IL-6 production wasa consequence of a specific IL-17A neutralization, the cells wereincubated with the Fyn SH3 wt domain (Grabulovski et al. (2007) JBC,282, p. 3196-3204) as a protein of irrelevant binding specificity inpresence of IL-17A (FIG. 13 (c)). As expected, no inhibition of IL-6production was observed, whereas clone 2C1 (SEQ ID NO: 110) was capableof inhibiting IL-17A-induced 11-6 production. In FIG. 13(d) the XTTassay is shown, confirming that all cells were viable after incubationwith Fyn SH3-derived IL-17A-binding polypeptides of the invention (at aconcentration of 750 nM) and IL-17 receptor (10 nM) for 24 hours.

Example 2.4 Stability

A crucial aspect of any biological compound intended for therapeuticapplications is its stability and resistance to aggregation when storedin solution. Fyn SH3-derived IL-17A-binding polypeptides of theinvention are particularly useful drug and diagnostic candidates becausethey have proven stable when stored at 4° C. or at −20° C. for at least6 months in simple phosphate-buffered saline.

Methods

Protein solutions of the IL-17A-binding polypeptides of the inventionwere stored for 6 months at 4° C. and at −20° C. after purification. Inorder to analyze protein stability and aggregation state, the proteinsolutions were filtered (Millex GP, 0.22 μm, Millipore) and sizeexclusion chromatography (SEC) was performed on an ÄKTA FPLC systemusing a Superdex 75 Short Column (5/150) (GE Healthcare)

Results

Fyn SH3-derived IL-17A-binding polypeptide G3 (SEQ ID NO: 37) wasproduced with an expression yield of 123 mg/L and eluted mainly assingle peak from the size exclusion chromatography column (see FIG. 14).

The stability and aggregation resistance of G3 (SEQ ID NO: 37) wasassessed by storing the protein at 4° C. and −20° C. in PBS. After 6months the status of the protein was examined by size exclusionchromatography. The measurements did not reveal any sign of aggregationor degradation. The elution profiles after 6 months of storage are shownin FIG. 15.

Example 2.5 In Vivo Half-Life

The in vivo half-life of the fusion protein of the invention E4-Fc (SEQID NO: 120) was determined by measuring E4-Fc (SEQ ID NO: 120)concentrations in mouse serum at different time points after a singlei.v. injection by ELISA.

Methods

Cloning and expression of E4-Fc (SEQ ID NO: 120) is described in Example2.2. 200 μl of a 3.3 μM (0.22 mg/ml) solution of E4-Fc (SEQ ID NO: 120)was injected i.v. into 5 mice (C57BL/6, Charles River). After 7 minutes,20 minutes, 1, 2, 4, 8, 24 and 48 h about 20 μl of blood were taken fromthe vena saphena with the capillary Microvette CB 300 (Sarstedt). Theblood samples were centrifuged for 10 min at 9500×g and the serum wasstored at −20° until ELISA analysis was performed. Using an E4-Fc (SEQID NO: 120) dilution series with known concentrations, the E4-Fc (SEQ IDNO: 120) concentration in serum was determined by ELISA: 50 μl ofbiotinylated IL-17A (30 nM) (R&D Systems, biotinylated usingNHS-PEO4-biotin (Pierce) according to the menu-facturer's instructions)were added to streptavidin-coated wells (Reactibind, Pierce) and afterblocking with PBS, 4% milk (Rapilait, Migros, Switzerland), 45 μl ofPBS, 4% milk and 5 μl of serum sample were added. After incubation for 1h and washing, bound Fc fusion proteins were detected with protein A-HRPconjugate (Sigma). Peroxidase activity was detected by addition ofQuantaRed enhanced chemifluorescent HRP substrate (Pierce). Fluorescenceintensity was measured after 5 to 10 min at 544 nm (excitation) and 590nm (emission). From the concentrations of E4-Fc (SEQ ID NO: 120)determined in serum (n≧3 per time point, except last time point: n=1) atdifferent time points and the resulting slope k of the elimination phase(plotted in a semi-logarithmic scale) the half-life of E4-Fc (SEQ ID NO:120) was calculated using to the formula t^(1/2)=ln 2/−k.

Results

The half-life of fusion protein of the invention E4-Fc (SEQ ID NO: 120)as calculated from the elimination phase (beta phase, 4 last timepoints) was 50.6 hours (see FIG. 16).

Example 2.6 ELISA for Determining the Binding Specificity ofIL-17A-Binding Polypeptides and Fusion Proteins

Methods

Target proteins human IL-17F (R&D systems), murine IL-17A (R&D Systems),human TNF-alpha (Thermo Scientific), human IL-6 (R&D Systems), bovineserum albumin (Sigma) and ovalbumin (Sigma) were coated on a MaxiSorpplate (Nunc) overnight (100 μl of each target at a concentration of 5μg/ml). Wells were washed three times with PBS and after blocking with200 μl of PBS, 4% Milk (Rapilait, Migros) and a washing step with PBS(as above), 50 μl of 2C1 (SEQ ID No: 110) at a final concentration of 50nM were added to the wells together with 50 μl of an anti-myc antibody(9E10, produced in-house, a stock solution of OD=2 and diluted 1:250 inPBS, 2% milk). After incubation the wells were washed three times withPBS and 100 μl of anti-mouse-HRP immunoconjugate (Sigma) diluted 1:1000in PBS, 2% milk were added to the wells. The 96-well plate was incubatedfor 1 h at RT and then washed three times with PBS, 0.1% Tween followedby three washes with PBS only. Colorimetric detection was done byaddition of 100 μl of BM blue POD substrate (Roche) and the reaction wasstopped with 60 μl 1 M H₂SO₄.

Results

Clone 2C1 (SEQ ID NO: 110) bound human IL-17A in a highly specificmanner and did not cross-react with any of the other tested proteins asshown by ELISA (FIG. 17). A small signal above background was observedfor IL-17F, but when 2C1 was probed to a IL-17F coated BIAcore chip, nodetectable binding was determined (data not shown).

Example 2.7 Fyn SH3-Derived Polypeptide of the Invention BindsSpecifically and with High Affinity to Human and Cynomolgus IL-17a

Methods

a) Specificity

For the determination of the binding specificity of IL-17A-bindingpolypeptides of the invention, the following target proteins were used(more target proteins compared to Example 2.6):

-   -   human IL-17A (R & D Systems)    -   human IL-17B (Peprotech)    -   human IL-17C(R & D Systems)    -   human IL-17D (Peprotech)    -   human IL-17E (Peprotech)    -   human IL-17F (Abd Serotec)    -   mouse IL-17A (R & D Systems)    -   rat IL-17A (Akron Biotech)    -   canine IL-17A (R & D Systems)    -   cynomolgus (macaca fascicularis) IL-17A (produced in-house in E.        coli, without signal peptide, with a C-terminal glycine residue        followed by a hexa-his tag, refolded from inclusion bodies, SEQ        ID NO: 132)    -   extra domain B of fibronectin (produced in-house, E. coli; see        Carnemolla et al. (1996) Int J Cancer, 68(3), p. 397-405)    -   Human IL-6 (R & D Systems)    -   Human TNF alpha (Thermo Scientific)    -   Ovalbumin (Sigma)    -   BSA (Sigma)

The target proteins were coated on a MaxiSorp plate (Nunc) overnight(100 μl of each target at a concentration of 10 μg/ml). Wells werewashed three times with PBS and after blocking with 200 μl of PBS, 4%Milk (Rapilait, Migros) for 1 hour at room temperature and a subsequentwashing step with PBS (as above), 50 μl of the Fyn SH3-derivedpolypeptide of the invention 2C1 (SEQ ID No: 110) at a finalconcentration of 80 nM were added to the wells together with 50 μl ofanti-myc antibody 9E10 (produced in-house, a stock solution of OD=2 anddiluted 1:250 in PBS, 2% milk). After incubation the wells were washedthree times with PBS and 100 μl of anti-mouse-HRP immunoconjugate(Sigma) diluted 1:1000 in PBS, 2% milk were added to the wells. The96-well plate was incubated for 1 h at RT and then washed three timeswith PBS, 0.1% Tween followed by three washes with PBS only.Colorimetric detection was done by addition of 100 μl of BM blue PODsubstrate (Roche) and the reaction was stopped with 60 μl 1 M H₂SO₄.

b) Affinity Measurements to Cynomolgus IL-17a

Affinity measurements were performed using a BIAcore 3000 instrument(Biacore). For the interaction analysis between cynomolgus IL-17A andthe Fyn SH3-derived polypeptide of the invention 2C1 (SEQ ID NO: 110) aCM5 chip (Biacore) was coated with 6900 RU cynomolgus IL-17A. Therunning buffer was HBS-EP (Biacore). The interactions were measured at aflow of 20 μl/min and injection of different concentrations of FynSH3-derived IL-17A-binding polypeptide of the invention 2C1 (SEQ ID NO:110). All kinetic data of the interaction (separate kon/koff) wereevaluated using BIA evaluation 3.2RC1 software.

Results

Fyn SH3-derived polypeptide of the invention 2C1 (SEQ ID NO: 110) boundhuman and cynomolgus IL-17A in a highly specific manner and did notcross-react with any of the other tested proteins as shown by ELISA(FIG. 18).

The affinity of monomeric Fyn SH3-derived polypeptide of the invention2C1 (SEQ ID NO: 110) for cynomolgus IL-17A was measured with Biacoreusing cynomolgus IL-17A produced in E. coli (refolded from inclusionbodies). 2C1 was found to bind cynomolgus IL-17A with a K_(D) of 11 nM(FIG. 19).

Example 2.8 Expression of Fyn SH3-Derived Polypeptides of the InventionFused to an Fc Part and to a Modified Fc Part of a Human IgG1 Antibodyin Mammalian Cells

The Fyn SH3-derived polypeptide of the invention 2C1 (SEQ ID NO: 110)was genetically fused to the Fc part of IgG1 (2C1-Fc, SEQ ID NO: 133)and expressed in HEK EBNA cells. The Fyn SH3-derived polypeptide of theinvention 2C1 (SEQ ID NO: 110) was also cloned as genetic fusion to themodified Fc part of human IgG1, comprising mutations L234A (alanineinstead of leucine at amino acid position 234) and L235A and expressedin HEK EBNA cells (2C1-Fc(LALA), SEQ ID NO: 134). Furthermore, thefollowing four variants of 2C1-Fc(LALA) fusion protein with differentlinker length between the Fyn SH3-derived polypeptide of the inventionand the Fc part were produced:

-   -   (SEQ ID NO: 135) “2C1-m5E-Fc(LALA)”; extension of hinge region        by 5 amino acids: EPKSS linker    -   (SEQ ID NO: 136) “2C1-m5-Fc(LALA)”; 5 amino acids extension,        GGGGS linker    -   (SEQ ID NO: 137) “2C1-m10-Fc(LALA)”; 10 amino acids extension,        GGGGS)₂ linker    -   (SEQ ID NO: 138) “2C1-m15-Fc(LALA)”; 15 amino acids extension,        (GGGGS)₃ linker        Methods        Cloning of “2C1-Fc”: Fyn SH3-Derived Polypeptide of the        Invention 2C1 (SEQ ID NO: 110) Fused to an Fc Part of a Human        IgG1 Antibody (SEQ ID NO: 133):

The gene encoding clone 2C1 (SEQ ID NO: 110) was used as a template andamplified using the primers SB3 (5′ CGA ATT CGG GAG TGA CAC TCT TTG TGGCCC 3′, SEQ ID NO: 139) and SB4 (5′ GAA GAT CTC TGG ATA GAG TCA ACT GGAGCC 3′, SEQ ID NO: 140) introducing the restriction sites EcoRI andBgIII. Obtained PCR product was digested with EcoRI and BgIII and clonedinto the previously double-digested pFUSE-hIgG1-Fc2 vector (Invivogen).For cloning this Fc fusion into the pCEP4 vector (Invitrogen), theresulting pFUSE vector containing the gene encoding the 2C1-Fc fusionwas used as template and amplified with the primers SB5 (5′ CCC AAG CTTGGG ATG GGC TAC AGG ATG CAA CTC CTG TC 3′, SEQ ID NO: 141) and SB6 (5′CGG GAT CCT CAT TTA CCC GGA GAC AGG GAG 3′, SEQ ID NO: 142), introducingHindIII and BamHI restriction sites. After digestion with HindIII/BamHI,the insert was ligated with previously double-digested pCEP4 vector,yielding the plasmid containing the genetic information of SEQ ID NO:133.

Cloning of “2C1-Fc(LALA)”: Fyn SH3-Derived Polypeptide of the Invention2C1 (SEQ ID NO: 110) Fused to a Modified Fc Part of a Human IgG1Antibody (L234A, L235A) (Yielding SEQ ID NO: 134)

The above mentioned plasmid containing the genetic information of 2C1-Fc(SEQ ID NO: 133) was used as a template for two PCR reactions. In thefirst reaction, the primers SB5 and SB7 (5′ ACT GAC GGT CCC CCC GCG GCTTCA GGT GCT GGG CAC 3′, SEQ ID NO: 143) were used. In the second PCR theprimers SB8 (5′ GCC GCG GGG GGA CCG TCA GTC TTC CTC TTC CC 3′, SEQ IDNO: 144) and SB6 were used. A PCR assembly with both fragments astemplates was performed, the resulting PCR product was digested withBamHI and HindIII and ligated with the digested pCEP4 vector asdescribed above.

Cloning of “2C1-m5E-Fc(LALA)” (SEQ ID NO: 135): Fyn SH3-DerivedPolypeptide of the Invention 2C1 (SEQ ID NO: 107) Fused with a 5 AminoAcid Linker EPKSS to a Modified Fc Part of a Human IgG1 Antibody (L234A,L235A)

The above mentioned plasmid containing the genetic information of2C1-Fc(LALA) (SEQ ID NO: 134) was used as a template for two PCRs. Inthe first reaction the primers SB5 and “Ba_2C1_R_EPKSS” (5′ GCT GCT TTTCGG TTC CTG GAT AGA GTC AAC TGG AGC CAC 3′, SEQ ID NO: 145) were used.In the second reaction the primers SB6 and “Ba_Hinge_F_EPKSS” (5′ GAACCG AAA AGC AGC GAC AAA ACT CAC ACA TGC CCA CCG 3′, SEQ ID NO: 146) wereused. A PCR assembly with both fragments as templates was performed, theresulting PCR product was digested with BamHI and HindIII and ligatedwith the digested pCEP4 vector as described above.

Cloning of “2C1-m5-Fc(LALA)” (SEQ ID NO: 136): Fyn SH3-DerivedPolypeptide of the Invention 2C1 (SEQ ID NO: 110) Fused with a 5 AminoAcid Linker GGGGS to a Modified Fc Part of a Human IgG1 Antibody (L234A,L235A)

The above mentioned plasmid containing the genetic information of2C1-Fc(LALA) (SEQ ID NO: 134) was used as a template for two PCRs. Inthe first reaction the primers SB5 and 47c.fo (5′ TGA ACC GCC TCC ACCCTG GAT AGA GTC AAC TGG AGC CAC 3′, SEQ ID NO: 147) were used. In thesecond reaction the primers SB6 and “Ba_Hinge_F_5aaGS-linker” (5′ GGTGGA GGC GGT TCA GAC AAA ACT CAC ACA TGC CCA CCG 3′, SEQ ID NO: 148) wereused. A PCR assembly with both fragments as templates was performed, theresulting PCR product was digested with BamHI and HindIII and ligatedwith the digested pCEP4 vector as described above.

Cloning of “2C1-m10-Fc(LALA)” (SEQ ID NO: 137): Fyn SH3-DerivedPolypeptide of the Invention 2C1 (SEQ ID NO: 110) Fused with a 10 AminoAcid Linker (GGGGS)₂ to a Modified Fc Part of a Human IgG1 Antibody(L234A, L235A)

The above mentioned plasmid containing the genetic information of2C1-Fc(LALA) (SEQ ID NO: 134) was used as a template for two PCRs. Inthe first reaction the primers SB5 and 47b.fo (5′ AGA GCC ACC TCC GCCTGA ACC GCC TCC ACC CTG GAT AGA GTC AAC TGG AGC CAC 3′, SEQ ID NO: 149)were used. In the second reaction the primers SB6 and“Ba_Hinge_F_10aaGS-linker” (5′ GGT GGA GGC GGT TCA GGC GGA GGT GGC TCTGAC AAA ACT CAC ACA TGC CCA CCG 3′, SEQ ID NO: 150) were used. A PCRassembly with both fragments as templates was performed, the resultingPCR product was digested with BamHI and HindIII and ligated with thedigested pCEP4 vector as described above.

Cloning of “2C1-m15-Fc(LALA)” (SEQ ID NO: 138): Fyn SH3-DerivedPolypeptide of the Invention 2C1 (SEQ ID NO: 110) Fused with a 15 AminoAcid Linker (GGGGS)₃ to a Modified Fc Part of a Human IgG1 Antibody(L234A, L235A)

The above mentioned plasmid containing the genetic information of2C1-Fc(LALA) (SEQ ID NO: 134) was used as a template for two PCRs. Inthe first reaction the primers SB5 and 47.fo.corr (5′ TGA TCC GCC ACCGCC AGA GCC ACC TCC GCC TGA ACC GCC TCC ACC CTG GAT AGA GTC AAC TGG AGCCAC 3′, SEQ ID NO: 151) were used. In the second reaction the primersSB6 and “Ba_Hinge_F_15aaGS-linker” (5′ GGT GGA GGC GGT TCA GGC GGA GGTGGC TCT GGC GGT GGC GGA TCA GAC AAA ACT CAC ACA TGC CCA CCG 3′, SEQ IDNO: 152) were used. A PCR assembly with both fragments as templates wasperformed, the resulting PCR product was digested with BamHI and HindIIIand ligated with the digested pCEP4 vector as described above.

For expression of the fusion proteins, the corresponding plasmids werepurified using an endotoxin free Megaprep kit (Qiagen) and used fortransient transfection of HEK EBNA cells (ATCC No CRL-10852). HEK EBNAcells were seeded at 30% confluence 24 hours prior to transfection. Themedium was replaced with DMEM/5% FCS/penstrep (Invitrogen) immediatelyprior to transfection. 60 μg of DNA was used to transfect 150 cm² ofadherent cells. DNA and PEI (25 kDa from Polysciences) were mixed in a1:3 ratio and vortexed for 10 sec. Then, the DNA/PEI mixture wasincubated at RT for 10 minutes and subsequently added to the HEK EBNAcells. After 24 hours the medium was replaced withCD-CHO/HT/L-glutamine/Penstrep (Invitrogen) and incubated at 37° C. with5% CO₂. The cell culture supernatant was harvested after 96 hours.

For protein purification, the cell culture supernatant was applied to aprotein A-sepharose affinity column. Subsequently, the column was washedwith PBS followed by protein elution using 0.1 M glycine pH 2.7. Elutedprotein was then dialysed into PBS. If needed, a second purificationstep for removal of endotoxins with Triton-X114 was performed (Magalhaeset al. (2007) J Pharm Pharmaceut Sci, 10(3), p. 388-404).

Results

The Fyn SH3-derived Fc fusions of the invention could be expressed andpurified. FIG. 20 shows the SDS PAGE analysis of the Fc fusion proteins.

Example 2.9 Fyn SH3-Derived Polypeptides of the Invention are Stable inHuman Serum

Protein drugs should be stable in serum for a certain period of time, inorder to be able to elicit pharmacodynamic effects in patients. In thisexample, the serum stability of 2C1-Fc (SEQ ID NO: 133) was tested.

Methods

A solution of 3 ml of human serum (Sigma) containing 10 μg/ml 2C1-Fc(SEQ ID NO: 133) was prepared and placed in an incubator at 37° C. 200μl samples were removed at indicated time points and frozen at −20° C.until the end of the experiment. After 5 days, an ELISA was performedwith the collected samples, using a 2C1-Fc sample (SEQ ID NO: 133) whichhas been stored at 4° C. in PBS as a control standard.

To perform the ELISA, IL-17A (R&D Systems) was coated on a MaxiSorpplate (Nunc) overnight (100 μl of 5 μg/ml). Wells were washed threetimes with PBS and after blocking with 200 μl of PBS, 4% Milk (Rapilait,Migros) and a washing step with PBS (as above), 100 μl of the testsamples comprising 2C1-Fc (SEQ ID NO: 133) (at the indicatedconcentrations) diluted in PBS, 2% Milk were added. After incubation,the wells were washed three times with PBS, followed by addition of 100μl Protein A-HRP (Sigma) diluted 1:1000 in PBS, 2% milk. The 96-wellplate was incubated for 1 h at RT and then washed three times with PBS,0.1% Tween followed by three washes with PBS only. Colorimetricdetection was done by addition of 100 μl of BM blue POD substrate(Roche) and the reaction was stopped with 60 μl 1 M H₂SO₄.

Results

After a 5-day storage period in human serum at 37° C. 2C1-Fc (SEQ ID NO:133) was able to bind its target IL-17A essentially as well as 2C1-Fc(SEQ ID NO: 133) which was stored in PBS at 4° C., indicating that2C1-Fc (SEQ ID NO: 133) is stable in human serum at 37° C. (FIG. 21).

Example 2.10 Fyn SH3-Derived Polypeptides of the Invention InhibitIL-17a In Vitro

In this assay the indicated Fyn SH3-derived polypeptides of theinvention were tested for their ability to inhibit IL-17A in vitro. Thecell assay is similar to the cell assay described in Example 2.3 of thisinvention, with the main exception that IL-17A is used at a lowconcentration of 1 ng/ml (compared to 50 ng/ml in Example 2.3) togetherwith TNF alpha (50 μg/ml).

Methods

Endotoxin levels of tested Fyn SH3-derived IL-17A-binding polypeptidesof the invention were less than 0.1 EU/ml, as determined by the Limulusamebocyte lysate (LAL) test (PYROGENT Single test Gel Clot LAL Assay(Lonza)).

Normal human dermal fibroblasts (NHDF, PromoCell Inc., NHDF-c, C12300)are used for the IL-17A inhibition cell assay. Addition of human IL-17A(R&D Systems) in combination with human tumor necrosis factor-α (TNF-α,Thermo Fisher Scientific) to the cell culture medium induces IL-6production by NHDF cells in a dose-dependent manner. IL-6 released intothe cell culture medium (PromoCell, C-23010) is quantified in cellculture supernatant by ELISA using a commercially available ELISA kit(R&D Systems, DuoSet ELISA System kit (DY206)).

10⁴ Normal Human Dermal Fibroblasts (PromoCell, NHDF-c, C12300) weredistributed per well (24 well plate, Nunc or TPP) and cultured for 24hours at 37° C. (medium: Fibroblast Growth Medium C-23010, PromoCell).The supernatant was aspirated and after mixing different concentrationsof Fyn SH3 derived IL-17A-binding polypeptides of the invention orIL-17A receptor Fc chimera (RnD Systems) with IL-17A (RnD Systems) andTNF alpha (Thermo Scientific) containing medium (1 ng/ml final IL-17Aconcentration and 50 μg/ml TNF alpha), 350 μl of the correspondingsolution was added per well, in triplicate (mixing ratio betweeninhibitor solution and cytokine-containing medium was 1:23). Controlwells included incubation without Fyn SH3-derived polypeptides (PBSonly), IL-17A alone, TNF-α alone and medium only. After 24 hoursincubation at 37° C. the supernatant was aspirated and the ELISAabsorbance (correlating to the IL-6 concentration) was determined byELISA according to the manufacturer's instructions (IL-6 ELISA kit, R&DSystems).

Results

NHDF cells were incubated with a constant concentration of IL-17A (1ng/ml) and TNF alpha (50 μg/ml) and with different concentrations of thecommercially available IL-17A receptor-Fc chimera or with differentconcentrations of the following Fyn SH3-derived polypeptides of theinvention:

-   -   2C1 (SEQ ID NO: 110)    -   2C1-Fc (SEQ ID NO:133)    -   2C1-Fc(LALA) (SEQ ID NO: 134)    -   2C1-m5E-Fc(LALA) (SEQ ID NO: 135)    -   2C1-m5-Fc(LALA) (SEQ ID NO: 136)    -   2C1-m10-Fc(LALA) (SEQ ID NO: 137)    -   2C1-m15-Fc(LALA) (SEQ ID NO: 138)

Table 3 shows the average of the IC₅₀ values obtained from several cellassays performed with the indicated Fyn SH3-derived polypeptides of theinvention. The best IC₅₀ value (0.11 nM) was obtained with2C1-m15-Fc(LALA) (SEQ ID NO: 138).

TABLE 3 Average IC₅₀ values of Fyn SH3-derived polypeptides of theinvention obtained from several cell assays. IC₅₀ value Standard Numberof (nM) Deviation cell assays 2C1 (SEQ ID NO: 110) 2.31 0.08 3 2C1-Fc(SEQ ID NO: 133) 1.13 0.30 4 2C1-Fc(LALA) (SEQ ID NO: 134) 1.09 0.53 42C1-m5E-Fc(LALA) 0.72 0.30 4 (SEQ ID NO: 135) 2C1-m5-Fc(LALA) 1.45 n.d.2 (SEQ ID NO: 136) 2C1-m10-Fc(LALA) 0.27 0.13 6 (SEQ ID NO: 137)2C1-m15-Fc(LALA) 0.11 0.02 3 (SEQ ID NO: 138) IL-17A-Receptor Fc chimera0.61 0.38 6 (R&D Systems)

Example 2.11 In Vivo Half-Life of 2C1-Fc(LALA) (SEQ ID NO: 134)

The in vivo half-life of the fusion protein of the invention2C1-Fc(LALA) (SEQ ID NO: 134) was determined by measuring 2C1-Fc(LALA)(SEQ ID NO: 134) concentrations in mouse serum at different time pointsafter a single i.v. injection.

Methods

2C1-Fc(LALA) (SEQ ID NO: 134) solution (0.2 mg/ml) was injected i.v.into 5 mice (C57BL/6, Charles River), 200 μl per mouse. After indicatedtime-points about 20 μl of blood were taken from the vena saphena withthe capillary Microvette CB 300 (Sarstedt). The blood samples werecentrifuged for 10 min at 9500×g and the serum was stored at −20° untilELISA analysis was performed. Using a 2C1-Fc(LALA) (SEQ ID NO: 134)dilution series with known concentrations, the 2C1-Fc(LALA) (SEQ ID NO:134) concentration in serum was determined by ELISA: 50 μl ofbiotinylated IL-17A (30 nM) (R&D Systems, biotinylated usingNHS-PEO4-biotin (Pierce) according to the manufacturer's instructions)were added to streptavidin-coated wells (Reactibind, Pierce) and afterblocking with PBS, 4% milk (Rapilait, Migros, Switzerland), 45 μl ofPBS, 4% milk and 5 μl of serum sample were added. After incubation for 1h and washing, bound Fc fusion proteins were detected with protein A-HRPconjugate (Sigma). Peroxidase activity was detected by addition ofQuantaRed enhanced chemifluorescent HRP substrate (Pierce). Fluorescenceintensity was measured after 5 to 10 min at 544 nm (excitation) and 590nm (emission). From the concentrations of 2C1-Fc(LALA) (SEQ ID NO: 134)determined in serum (mouse number n=5 per time point) at different timepoints and the resulting slope k of the elimination phase (plotted in asemi-logarithmic scale) the half-life of 2C1-Fc(LALA) (SEQ ID NO: 134)was calculated using to the formula t^(1/2)=ln 2/−k.

Results

The half-life of fusion protein of the invention 2C1-Fc(LALA) (SEQ IDNO: 134) as calculated from the elimination phase (beta phase, 4 lasttime points) was 53 hours (see FIG. 22).

Example 2.12 Fyn SH3-Derived Polypeptides of the Invention NeutralizeHuman IL-17a In Vivo

Human IL-17A is able to bind and stimulate the mouse IL-17 receptor,leading to an elevation and subsequent secretion of mouse KC (CXCL1)chemokine (Allan B. et al. (2007) WO2007/070750 of Eli Lilly, US). Theobserved KC levels 2 hours after s.c. IL-17A injection (3 μg) werebetween 500 and 1000 μg/ml in the serum, compared to around 100 μg/ml KCbasal levels.

Methods

a) In Vivo Neutralization of IL-17a Using Monomeric Fyn SH3 DerivedPolypeptide of the Invention 2C1 (SEQ ID NO: 110)

Fyn SH3-derived polypeptide of the invention 2C1 (SEQ ID NO: 110) (17μg) was co-injected (s.c.) with 3 μg of human IL-17A (R&D Systems) intoC57BL/6 mice, and 2 hours after injection, blood samples were taken fromthe vena saphena with the capillary Microvette CB 300 (Sarstedt). Theblood samples were centrifuged for 10 min at 9500×g and the serum wasstored at −20° until ELISA analysis was performed. KC levels in serumwere determined using the commercially available Quantikine mouseCLCL1/KC kit (R&D Systems). Control groups included mice injected withIL-17A and the Fyn SH3 wt domain (see Grabulovski et al. (2007) JBC,282, p. 3196-3204) as a protein of irrelevant binding specificity, PBSonly, IL-17A only, only Fyn SH3-derived polypeptide of the invention 2C1(SEQ ID NO: 110) or mice given Fyn SH3 wt protein only.

b) In Vivo Neutralization Using the 2C1-Fc Fusion (SEQ ID NO: 133):

Fyn SH3-derived polypeptide of the invention 2C1-Fc (SEQ ID NO: 133) (44μg/mouse) was injected i.v. into C57BL/6 mice. After 20-60 minutes, 3μg/mouse of human IL-17A (R&D Systems) was injected s.c. and 2 hoursafter IL-17A injection, blood samples were taken from the vena saphenawith the capillary Microvette CB 300 (Sarstedt). The blood samples werecentrifuged for 10 min at 9500×g and the serum was stored at −20° untilELISA analysis was performed. KC levels in serum were determined usingthe commercially available Quantikine mouse CLCL1/KC kit (R&D Systems).Control groups included mice injected with PBS (i.v.) and IL-17A (s.c.),PBS only (i.v. and s.c.), and Fyn SH3-derived polypeptide of theinvention 2C1-Fc (SEQ ID NO: 133) i.v. followed by PBS (s.c.).

Results

After s.c. injection of human IL-17A into mice the animals overexpress achemokine called KC. Elevated KC levels in the sera of mice can bemeasured by ELISA. Injection of a Fyn SH3-derived polypeptide of theinvention prevented the up-regulation of KC.

a)

IL-17A and monomeric Fyn SH3-derived polypeptide 2C1 (SEQ ID NO: 110) ofthe invention were co-injected s.c. into mice (C57BL/6). Because of theinhibitory properties of the Fyn SH3-derived polypeptide of theinvention 2C1 (SEQ ID NO: 110), KC levels were not elevated in thisgroup, they remained low, almost comparable to basal levels. In order todemonstrate that inhibition of KC production was due to specific IL-17Aneutralization, mice were co-injected with IL-17A and the wild-type FynSH3 domain (which has no binding affinity to IL-17A); in these mice, KClevels were as high as in the group receiving IL-17A only. FIG. 23 showsthe results obtained from this experiment.

b)

In this second acute inflammation experiment, the Fyn SH3-derivedpolypeptide of the invention 2C1-Fc (SEQ ID NO: 133) was injected i.v.,followed by s.c. injection of IL-17A. As above in a), the FynSH3-derived polypeptide of the invention prevented the up-regulation ofKC levels in the serum. FIG. 24 shows the inhibition of IL-17A by 2C1-Fc(SEQ ID NO: 133) in vivo.

Example 3 Anti-Chymase Fyn SH3 Derivatives Example 3.1 Fyn SH3-DerivedPolypeptides of the Invention Bind to Chymase as Determined byMonoclonal ELISA Using Bacterial Lysate Supernatants Containing the FynSH3-Derived Polypeptides of the Invention

Methods:

DNA encoding the amino acid sequences shown in SEQ ID NOs: 153 to 166were cloned into the cytosolic expression vector pQE-12 with aC-terminal myc and hexa his tag. After bacterial electroporation,bacterial lysates containing the Fyn SH3-derived polypeptides wereproduced as described in Bertschinger et al. (Bertschinger et al. (2007)Protein Eng Des Sel, 20(2), p. 57-68). Chymase was produced as describedin Perspicace et al. (Perspicace et al. (2009) J Biomol Screen, 14(4),p. 337-349). The protein was biotinylated according to themanufacturer's instructions using EZ-link sulfo-NHS-SS-biotin (Perbio)and finally contained 3 biotin molecules per chymase molecule. For theELISA experiment, biotinylated chymase was added to streptavidin-coatedwells (StreptaWells, High Bind, Roche) at a concentration of 100 nM andafter blocking with PBS, 2% milk (Rapilait, Migros, Switzerland), 40 μlof the bacterial supernatant containing the corresponding FynSH3-derived polypeptide were added to the wells together with 10 μl ofan anti-myc antibody (9E10, at a final concentration of 10 μg/ml in PBS,2% Milk). After incubating for 1 h and washing, detection was made withanti-mouse IgG HRP antibody conjugate (Sigma). Peroxidase activity wasdetected by adding BM blue POD substrate (Roche) and the reaction wasstopped by adding 1M H₂SO₄. The DNA sequence of the binders was verifiedby DNA sequencing (BigDye Terminator v3.1 cycle sequencing kit, ABIPRISM 3130 Genetic Analyzer, Applied Biosystems).

Results:

The amino acid sequences of Fyn SH3-derived chymase binders (asdetermined by phage ELISA) is presented in SEQ ID NOs: 153 to 166 asappended in the sequence listing. SEQ ID NOs: 2 to 15 read:

(E4) SEQ ID NO: 153  GVTLFVALYDYNATRWTDLSFHKGEKFQILEFGPGDWWEARSLTTGETGYIPSNYVAPVDSIQ (B5) SEQ ID NO: 154 GVTLFVALYDYNATRWTDLSFHKGEKFQILDGDSGDWWEARSLTTGE TGYIPSNYVAPVDSIQ (A4)SEQ ID NO: 155  GVTLFVALYDYQADRWTDLSFHKGEKFQILDASPPGDWWEARSLTTGETGYIPSNYVAPVDSIQ (F12) SEQ ID NO: 156 GVTLFVALYDYRAERSTDLSFHKGEKFQILDMTVPNGDWWEARSLTT GETGYIPSNYVAPVDSIQ(G2.3) SEQ ID NO: 157  GVTLFVALYDYNATRWTDLSFHKGEKFQILDWTTANGDWWEARSLTTGETGYIPSNYVAPVDSIQ (D7) SEQ ID NO: 158 GVTLFVALYDYQADRWTDLSFHKGEKFQILSFHVGDWWEARSLTTGE TGYIPSNYVAPVDSIQ (H2)SEQ ID NO: 159  GVTLFVALYDYQADRWTDLSFHKGEKFQILRFDIGDWWEARSLTTGETGYIPSNYVAPVDSIQ (E3) SEQ ID NO: 160 GVTLFVALYDYQADRWTDLSFHKGEKFQILNASGPGDWWEARSLTTG ETGYIPSNYVAPVDSIQ (D2)SEQ ID NO: 161  GVTLFVALYDYEAQTWHDLSFHKGEKFQILNSSEGEYWEARSLTTGETGLIPSNYVAPVDSIQ (H11) SEQ ID NO: 162 GVTLFVALYDYKAQRWTDLSFKGEKFQILQAHQKTGDWWEARSLTTG ETGLIPSNYVAPVDSIQ (B10)SEQ ID NO: 163  GVTLFVALYDYEALHWHQLSFHKGEKSQILNSSEGTYWEARSLTTGETGWIPSNYVAPGDSIQ (E5) SEQ ID NO: 164 GVTLFVALYDYKAQRWLDLSFHEGEKFQILSTDSGDWWEARSLTTGE TGYIPSNYVAPVDSIQ (C5)SEQ ID NO: 165  GVTLFVALYDYEAPTWLHLSFHKGEKFQILNSSEGPWWEARSLTTGETGFIPSNYVAPVDSIQ (A8) SEQ ID NO: 166 GVTLFVALYDYEAANWFQLSFHKGEKFQILNSSEGPLWEARSLTTGE TGGIPSNYVAPVDSIQ

Example 3.2 Purified Fyn SH3-Derived Polypeptides of the Invention BindSpecifically to Chymase as Determined by ELISA

Methods:

Fyn SH3-derived polypeptides (SEQ ID NO: 153-160) were expressed andpurified as described in Grabulovski et al. (Grabulovski et al. (2007)JBC, 282, p. 3196-3204). Biotinylated chymase or biotinylated bovineserum albumin (BSA) as an irrelevant target protein (Sigma;biotinylation was performed according to the manufacturer's instructionsusing EZ-link sulfo-NHS-SS-biotin (Perbio)) was added tostreptavidin-coated wells (StreptaWells, High Bind, Roche) at aconcentration of 100 nM and after blocking with PBS, 2% milk (Rapilait,Migros, Switzerland), 50 μl of the corresponding Fyn SH3-derivedpolypeptide at a final concentration of 200 nM were added to the wellstogether with 50 μl of an anti-myc antibody (9E10, at a finalconcentration of 5 μg/ml in PBS, 2% Milk). After incubating for 1 h andwashing, detection was made with anti-mouse IgG HRP antibody conjugate(Sigma). Peroxidase activity was detected by adding BM blue PODsubstrate (Roche) and the reaction was stopped by adding 1M H₂SO₄.

Results:

FIG. 25 shows the ELISA signals on chymase and BSA coated wells,indicating specific binding to chymase.

Example 3.3 Fyn SH3-Derived Polypeptides of the Invention are Monomericand do not Aggregate as Determined by Size Exclusion Chromatography

Methods

After purification of the Fyn SH3-derived polypeptides (SEQ ID NOs:153-160) as described in Example 3.2, size exclusion chromatography(SEC) was performed on an ÄKTA FPLC system using a Superdex 75 Column(5/150) (GE Healthcare).

Results

Size exclusion chromatography (SEC) profiles demonstrated that allselected constructs eluted mainly as single, monomeric peaks (see FIG.26).

Example 3.4 Fyn SH3-Derived Polypeptides of the Invention Bind with HighAffinity to Chymase as Determined by Surface Plasmon ResonanceExperiments

Methods:

Affinity measurements of selected Fyn SH3-derived polypeptides (SEQ IDNO: 153-160) were performed using a BIAcore 3000 instrument (Biacore).For the interaction analysis between biotinylated chymase and monomericFyn SH3-derived polypeptides, a streptavidin SA chip (Biacore) wasimmobilized with 1331 RU biotinylated chymase. The running buffer wasPBS, 0.005% Tween 20. The interactions were measured at a flow of 30μl/min and injections of different concentrations of Fyn SH3-derivedchymase-binding polypeptides. All kinetic data of the interaction(separate kon/koff) were evaluated using BIA evaluation 3.2RC1 software

Results:

The binding properties were analyzed by real-time interaction analysison a BIAcore chip revealing the following dissociation constants (KD)and k_(off) values for the Fyn SH3-derived polypeptides (Table 4):

TABLE 4 Dissociation konstants and k_(off) values of Fvn SH3-derivedpolypeptides. Clone SEQ ID NO: K_(D) (nM) k_(off) (s⁻¹) F12 156 36.0 2.3× 10⁻³ G2.3 157 14.0 8.2 × 10⁻³ B5 154 5.0 3.6 × 10⁻³ D7 158 15.0 1.1 ×10⁻² E3 160 13.0 9.3 × 10⁻³ H2 159 32.0 2.1 × 10⁻³ A4 155 2.0 2.0 × 10⁻³E4 153 0.9 6.6 × 10⁻⁴

Example 3.5 Fyn SH3-Derived Polypeptides of the Invention InhibitProtease Activity of Chymase

The MR121 peptide fluorescence assay described below is based on thefact that MR121 forms a non-fluorescent ground state complex withtryptophan. In solution this formation occurs at millimolarconcentrations of tryptophan. Here, the substrate peptide is labeled atone terminus with tryptophan and at the other terminus with thefluorophore MR121. In absence of protease activity, the substrateremains intact and the MR121 fluorescence is reduced by the high localconcentration of tryptophan. If the substrate is cleaved by chymase, theMR121 fluorescence can be recorded. Therefore, the enzymatic reactioncan be followed in a kinetic measurement detecting an increase of MR121fluorescence during the reaction time. Calculating the slope in thelinear range of the kinetic provides the value for the activity of theenzyme.

Methods:

The chymase fluorescent substrate kinetic assay was performed intriplicate at room temperature in 96-well microtiter plates (Costar).Each well contained 100 μl assay buffer (100 mM Hepes, pH 7.4; 0.01%Triton X-100, 80 μg/ml heparin) with 1 nM chymase, 1 μM unlabeled and100 nM MR121 peptide (MR121-CAAPFW; Biosyntan GmbH, Berlin). FynSH3-derived (SEQ ID NOs: 153-160) were serially diluted in assay buffer(100 mM Hepes, pH 7.4; 0.01% Triton X-100, 80 μg/ml heparin) and addedto the reaction solution as specified above. The enzymatic reaction wasfollowed in a plate reader (Tecan Ultra, Tecan) at 612 nm excitation and670 nm emission for 20 min in a kinetic measurement, detecting anincrease of MR121 fluorescence during the reaction time. The slope inthe linear range of the kinetic was calculated and IC₅₀ values of theFyn SH3-derived polypeptides were calculated using a four parameterequation for curve fitting.

Results:

The titrated Fyn SH3-derived polypeptides showed dose-response curvesdemonstrating that they are potent inhibitors of chymase activity (seeTable 5).

TABLE 5 IC₅₀ values for inhibition of chymase activity. Clone SEQ ID NO:IC₅₀ (nM) F12 156 5 G2.3 157 1 B5 154 11 D7 158 6 E3 160 78 H2 159 18 A4155 4 E4 154 2

Example 3.6 Crystal Structure of Chymase and Fyn SH3-DerivedPolypeptides of the Invention Reveals Blockade of the Catalytic Site ofChymase by Fyn SH3-Derived Polypeptides of the Invention

Three selected Fyn SH3-derived polypeptides, B5 (SEQ ID NO: 154), A4(SEQ ID NO: 155) and E4 (SEQ ID NO: 153) were co-crystallized withchymase.

Methods:

Prior to crystallization experiments the Fyn SH3-derivedpolypeptides-chymase complexes were concentrated to 15 mg/ml.Crystallization screening against an INDEX screen (Hampton Research) wasperformed at 21° C. either in sitting drops by vapor diffusion or inmicrobatch experiments. Crystals appeared within one day and grew totheir final size within 3 days after setup.

In all cases, data were processed with XDS (Kabsch W. (2010) ActaCrystallogr D Biol Crystallogr. (66) p. 125-132.) and scaled with SADABS(obtained from Bruker AXS). Refinement was performed with Refmac5(Murshudov G N, et al. (1997). Acta Crystallogr D Biol Crystallogr.,(53) p. 240-255) from the CCP4 suite (The CCP4 suite: programs forprotein crystallography. (1994) Acta Crystallogr D Biol Crystallogr.,(50), p. 760-763) or BUSTER (Bricogne G. (1993) Acta Crystallogr D BiolCrystallogr. (49), p. 37-60., Roversi P et al. (2000), Acta CrystallogrD Biol Crystallogr., (56) p. 1316-23, Blanc E. et al. (2004), ActaCrystallogr D Biol Crystallogr. (60) p. 2210-2221) and model buildingdone with COOT (Emsley P et al. (2004) Acta Crystallogr D BiolCrystallogr., (60), p. 2126-2132).

Results:

Three different Fyn SH3 derived polypeptides binding to chymase (B5 (SEQID NO: 154) A4 (SEQ ID NO: 155) and E4 (SEQ ID NO: 153)) wereco-crystallized with chymase.

TABLE 6 The chymase-Fyn-SH3 derived polypeptide A4 (SEQ ID NO: 155)complex: Crystal SG19 59.630 92.792 116.256 90 90 90 parametersResolution 1.51 Å Crystallization 0.1M Citric acid pH 3.5, 25% PEG 3′350buffer Data collection and Data were collected on beam line X10SA(PXIII) refinement at the Swiss Light Source (SLS) at wavelength 1.0 Åusing a Pilatus pixel detector. For 101765 unique reflections to 1.51 Åresolution the merging R-factor on intensities was 6.5%. The finalR-values were 18.9% (all data) and 21.5% (5% R-free).

TABLE 7 Contacts between chymase and Fyn SH3-derived polypeptide A4 (SEQID NO: 155) All atom-atom contacts <3.5 Å are tabulated. Duplicates mayoccur as some residues have alternate conformations. The Fynomernumbering was chosen so that the first residue well visible in the firstelectron density is numbered 2. The chymase sequence is numberedserially from 1, so the catalytic serine is 182. 49 contacts found:CHYMASE FYNOMER DISTANCE 201(SER) OG 13(ALA) C 3.45 13(ALA) O 3.3114(ASP) C 3.06 200(ARG) CA 14(ASP) O 3.30 201(SER) N 14(ASP) O 2.90201(SER) OG 14(ASP) O 3.33 15(ARG) N 3.19 199(GLY) O 15(ARG) CA 3.32201(SER) OG 15(ARG) C 3.09 15(ARG) O 2.97  83(THR) O 15(ARG) NH1 2.94 84(SER) O 15(ARG) NH1 3.14  86(LEU) CD1 15(ARG) NH1 3.49  83(THR) O15(ARG) NH2 3.26 199(GLY) O 16(TRP) N 2.86 179(LYS) CE 16(TRP) O 3.37199(GLY) N 16(TRP) CD2 3.39 182(SER) OG 16(TRP) NE1 3.32 199(GLY) CA16(TRP) CE3 3.42 199(GLY) N 16(TRP) CE3 3.26 199(GLY) C 16(TRP) CE3 3.45199(GLY) CA 16(TRP) CZ3 3.44 199(GLY) N 16(TRP) CZ3 3.37 177(ALA) O16(TRP) CZ3 3.40 16(TRP) CH2 3.45  77(ARG) NH1 31(ASP) OD2 2.82  77(ARG)NH2 33(SER) OG 3.08 34(PRO) CG 3.50  82(ASN) CA 35(PRO) O 3.31  83(THR)N 35(PRO) O 2.84  83(THR) OG1 35(PRO) O 3.46  81(TYR) O 35(PRO) CD 3.41 84(SER) OG 36(GLY) CA 3.48  83(THR) OG1 36(GLY) CA 3.31 36(GLY) C 3.48 84(SER) OG 37(ASP) N 2.73 37(ASP) CB 3.42 37(ASP) CG 3.15 37(ASP) OD13.43 37(ASP) OD2 3.44 158(ARG) NH1 37(ASP) OD2 2.98  83(THR) OG1 38(TRP)N 3.27 38(TRP) O 2.83  83(THR) O 38(TRP) CD1 3.27  28(LYS) NZ 40(GLU)OE2 2.78  22(THR) O 42(ARG) NH1 3.31  81(TYR) OH 51(TYR) CZ 3.45 81(TYR) CZ 51(TYR) OH 3.49  81(TYR) OH 51(TYR) OH 2.59

It may be clearly seen that Trp16 of A4 inserts into the primaryspecificity pocket of chymase, which is thus inhibited.

TABLE 8 The chymase-Fyn SH3-derived polypeptide E4 (SEQ ID NO: 153)complex: Crystal SG19 58.998 59.855 89.711 90 90 90 parametersResolution 1.4 Å Crystallization 0.1M Bis-Tris pH 5.5, 25% PEG 3′350buffer Data collection Data were collected on beam line X10SA (PXIII) atthe and Swiss Light Source (SLS) at wavelength 1.0 Å using a refinementPilatus pixel detector. For 63158 unique reflections to 1.4 Å resolutionthe merging R-factor on intensities was 9.9%. The final R- values were18.6% (all data) and 20.5% (5% R-free).

TABLE 9 Contacts between chymase and E4 (SEQ ID NO: 153) All atom-atomcontacts <3.5 Å are tabulated. Duplicates may occur as some residueshave alternate conformations. The Fynomer numbering was chosen so thatthe first residue well visible in the first electron density is numbered2. The chymase sequence is numbered serially from 1, so the catalyticserine is 182 (with closest contact 3.52 Å in this structure). In thisstructure the increased number of contacts occurs only because at thehigher resolution it was possible to assign more alternativeconformations to side chains, which are then counted twice. 75 contactsfound: CHYMASE FYNOMER DISTANCE 201(SER) OG 13(ALA) C 3.36 13(ALA) O3.23 14(THR) C 3.15 14(THR) O 3.49 200(ARG) CA 14(THR) O 3.42 201(SER) N14(THR) O 3.05 201(SER) OG 15(ARG) N 3.26 15(ARG) N 3.27 199(GLY) O15(ARG) CA 3.24 15(ARG) CA 3.24 201(SER) OG 15(ARG) C 3.20 199(GLY) O15(ARG) C 3.50 201(SER) OG 15(ARG) C 3.12 199(GLY) O 15(ARG) C 3.49201(SER) OG 15(ARG) O 3.05 15(ARG) O 2.84  84(SER) O 15(ARG) CZ 3.43 83(THR) O 15(ARG) NH1 3.33  84(SER) O 15(ARG) NH1 2.80  84(SER) O15(ARG) NH1 3.04  86(LEU) CG 15(ARG) NH1 3.35  84(SER) O 15(ARG) NH12.66  84(SER) O 15(ARG) NH1 2.85  83(THR) O 15(ARG) NH2 3.08  84(SER) O15(ARG) NH2 3.35  84(SER) O 15(ARG) NH2 3.38 159(ASP) OD2 15(ARG) NH22.71 199(GLY) O 16(TRP) N 2.83 179(LYS) NZ 16(TRP) O 2.82 179(LYS) CE16(TRP) O 3.43 199(GLY) N 16(TRP) CD2 3.43 178(PHE) CD1 16(TRP) CE3 3.44199(GLY) N 16(TRP) CE3 3.22 199(GLY) CA 16(TRP) CE3 3.42 199(GLY) N16(TRP) CZ3 3.35 199(GLY) CA 16(TRP) CZ3 3.44 177(ALA) O 16(TRP) CZ33.44 16(TRP) CH2 3.49  24(ASN) ND2 28(GLN) CB 3.50  24(ASN) OD1 28(GLN)CG 3.39  24(ASN) ND2 28(GLN) CG 3.23 28(GLN) CD 3.40 28(GLN) OE1 3.33 23(SER) OG 30(LEU) O 3.13  24(ASN) H 30(LEU) CD2 3.39  24(ASN) N30(LEU) CD2 3.44  77(ARG) NH1 31(GLU) OE1 2.84 31(GLU) OE2 3.42  77(ARG)NH2 31(GLU) OE2 2.96  83(THR) N 34(PRO) O 2.83  83(THR) OG1 34(PRO) O3.37  83(THR) CG2 34(PRO) O 3.49  82(ASN) CA 34(PRO) O 3.32  84(SER) OG36(ASP) N 3.48 36(ASP) CB 3.39  83(THR) OG1 37(TRP) N 3.17 37(TRP) C3.45  83(THR) CB 37(TRP) O 3.39  83(THR) OG1 37(TRP) O 2.68 37(TRP) CB3.46  84(SER) OG 37(TRP) CD1 3.43  28(LYS) CE 39(GLU) OE2 3.49  28(LYS)NZ 39(GLU) OE2 2.63  24(ASN) O 41(ARG) NE 3.05  24(ASN) O 41(ARG) NE2.63  24(ASN) O 41(ARG) CZ 3.38  24(ASN) O 41(ARG) CZ 3.29  24(ASN) O41(ARG) NH2 2.86  24(ASN) O 41(ARG) NH2 3.09  22(THR) OG1 41(ARG) NH22.95  26(PRO) O 41(ARG) NH2 2.59  83(THR) CG2 50(TYR) CE1 3.41  81(TYR)OH 50(TYR) CE2 3.37 50(TYR) CZ 3.33 50(TYR) OH 2.69

TABLE 10 The chymase-Fyn SH3-derived polypeptide B5 (SEQ ID NO: 154)complex: Crystal SG19 56.937 64.124 174.987 90 90 90 parametersResolution 1.8 Å Crystallization 0.15M DL-Malic acid pH 7.0, 20% PEG3′350 buffer Data collection Data were collected on beam line X10SA(PXIII) at the and Swiss Light Source (SLS) at wavelength 1.0 Å using arefinement Pilatus pixel detector. For 62210 unique reflections to 1.78Å resolution the merging R-factor on intensities was 9.4%. The final R-values were 18.0% (all data) and 21.2% (5% R-free).

TABLE 11 Contacts between chymase and B5 (SEQ ID NO: 154) All atom-atomcontacts <3.5 Å are tabulated. Duplicates may occur as some residueshave alternate conformations. The Fynomer numbering was chosen so thatthe first residue well visible in the first electron density is numbered2. The Chymase sequence is numbered serially from 1, so the catalyticserine is 182. In this structure the increased number of contacts occurspartly because Trp16 of B5 was assigned 2 alternative conformations andpartly due to slight differences in B5 Arg15. 67 contacts found: CHYMASEFYNOMER DISTANCE 201(SER) OG 13(ALA) C 3.28 13(ALA) O 3.34 13(ALA) CB3.33 14(THR) N 3.43 14(THR) C 3.11 200(ARG) CA 14(THR) O 3.45 201(SER) N14(THR) O 2.96 201(SER) OG 14(THR) O 3.44 15(ARG) N 3.18 199(GLY) O15(ARG) CA 3.14 201(SER) OG 15(ARG) C 3.26 15(ARG) O 3.17 198(TYR) OH15(ARG) NH1 3.43 198(TYR) CZ 15(ARG) NH1 3.38  85(THR) O 15(ARG) NH13.38 159(ASP) OD2 15(ARG) NH1 3.14 159(ASP) CG 15(ARG) NH2 3.41 159(ASP)OD2 15(ARG) NH2 3.16 159(ASP) OD1 15(ARG) NH2 2.88 199(GLY) O 16(TRP) N2.92 16(TRP) N 2.94 179(LYS) NZ 16(TRP) O 2.82 16(TRP) O 2.91 178(PHE)CD1 16(TRP) CD1 3.28 178(PHE) CE1 16(TRP) CD1 3.35 200(ARG) O 16(TRP)CD1 3.15 199(GLY) C 16(TRP) CD1 3.39 199(GLY) O 16(TRP) CD1 3.37199(GLY) N 16(TRP) CD2 3.40 16(TRP) NE1 3.06 199(GLY) CA 16(TRP) NE13.30 199(GLY) N 16(TRP) CE2 3.34 16(TRP) CE3 3.19 199(GLY) CA 16(TRP)CE3 3.36 178(PHE) CD1 16(TRP) CE3 3.43 177(ALA) O 16(TRP) CZ3 3.29199(GLY) N 16(TRP) CZ3 3.26 199(GLY) CA 16(TRP) CZ3 3.32 182(SER) OG16(TRP) CZ3 2.92 177(ALA) O 16(TRP) CH2 3.41 182(SER) OG 16(TRP) CH22.95  77(ARG) NH1 31(ASP) OD2 2.86  83(THR) N 34(SER) O 3.04  83(THR)OG1 34(SER) O 3.44  83(THR) CG2 34(SER) O 3.37  77(ARG) NH2 34(SER) CB3.44  77(ARG) CZ 34(SER) OG 3.36  77(ARG) NH1 34(SER) OG 3.27  77(ARG)NH2 34(SER) OG 2.67  83(THR) OG1 35(GLY) CA 3.23 35(GLY) C 3.30  84(SER)OG 36(ASP) N 3.35  83(THR) OG1 37(TRP) N 3.41 37(TRP) C 3.49  83(THR) CB37(TRP) O 3.21  83(THR) OG1 37(TRP) O 2.55  84(SER) OG 37(TRP) CD1 3.48 28(LYS) NZ 39(GLU) CD 3.43 39(GLU) OE2 2.53  25(GLY) CA 41(ARG) CZ 3.33 26(PRO) O 41(ARG) NH1 3.25  23(SER) O 41(ARG) NH2 3.07  25(GLY) N41(ARG) NH2 3.27  25(GLY) CA 41(ARG) NH2 3.38  81(TYR) OH 50(TYR) CZ3.40  45(HIS) CB 50(TYR) OH 3.41  81(TYR) OH 50(TYR) OH 2.71

From the solved structures it can be seen that the main element for theinteraction between the Fyn SH3-derived polypeptides and chymase are thesequence motif Arg15-Trp16 of the Fyn SH3-derived polypeptides, whichconfer to tight binding into the chymase active site. It is obvious thatsuch a binding in the active site prevents the enzyme from being active,thus explaining the potent IC₅₀ values which have been determined in theenzymatic assay (Example 3.5).

Other indicated amino acids of the Fyn SH3-derived polypeptides makeadditional surface contacts with the 24 loop of chymase.

All six complex structures are very similar. The slight differences inthe Fyn SH3-derived polypeptides-chymase orientation come from both thesequence differences and crystal packing and are approximately a rigidbody rotation about Trp16 in the S1 pocket of chymase. The presence of aFyn SH3-derived polypeptide has only a minor influence on the overallconformation of chymase. The most pronounced change affects the 24 loopof chymase which seems to adapt slightly upon binding.

All resolved Fyn SH3-derived polypeptides adopt a typical SH3 domainfold.

Example 4 Anti-HER2 Fyn SH3 Derivatives Example 4.1 Fyn SH3 DerivedPolypeptides Bind to HER2

Methods

1) Phage ELISA on Recombinant HER2 Protein

DNA encoding the amino acids shown in SEQ ID NOs: 175 to 287 were clonedinto the phagemid vector pHEN1 as described for the Fyn SH3 library inGrabulovski et al. (Grabulovski et al. (2007) JBC, 282, p. 3196-3204).Phage production was performed according to standard protocols (Viti, F.et al. (2000) Methods Enzymol. 326, 480-505). Monoclonal bacterialsupernatants containing phages were used for ELISA: biotinylatedextracellular domain of HER2 comprising amino acids 23-652 of thefull-length protein (purchased from Bender Medsystems, or from R&D asfusion to human Fcγ1; biotinylation was performed withsulfo-NHS-LC-biotin (Pierce) according to the manufacturer'sinstructions) was immobilized on streptavidin-coated wells(StreptaWells, High Bind, Roche), and after blocking with 2% milk(Rapilait, Migros, Switzerland) in PBS, 20 μl of 10% milk in PBS and 80μl of phage supernatants were applied. After incubation for 1 hr,unbound phage were washed off, and bound phages were detected withanti-M13-HRP antibody conjugate (GE Healthcare). The detection ofperoxidase activity was done by adding BM blue POD substrate (Roche) andthe reaction was stopped by adding 1 M H₂SO₄. The phage ELISA positiveclones were tested by phage ELISA for the absence of cross reactivity toStreptavidin (StreptaWells, High Bind, Roche) and to human IgG (Sigma).

The DNA sequence of the specific binders was verified by DNA sequencing.

2) FACS Experiment on HER2 Overexpressing SKOV-3 Cells

DNA encoding the polypeptides shown in SEQ ID NOs: 167 to 174 and SEQ IDNOs: 288-318 were subcloned into the bacterial expression vector pQE12so that the resulting constructs carried a C-terminal myc-hexahistidinetag (SEQ ID NO: 328) as described in Grabulovski et al. (Grabulovski etal. (2007) JBC, 282, p. 3196-3204). The polypeptides were expressed inthe cytosol of E. coli bacteria, and 1.8 ml of cleared lysate wasprepared per ml original culture. 100 μl cleared lysate containing thepolypeptides was mixed with 100 μl cell suspension containing 1.25×10⁵SKOV-3 cells in PBS/1% FCS/0.2% sodium azide. After 60 min incubation onice, cells were washed, and bound sequences were detected by 10 μg/mlanti-myc mouse antibody 9E10 (Roche), followed by anti-mouseIgG-Alexa488 conjugate (Invitrogen). The stained cells were thenanalyzed in a FACS analyzer. The DNA sequence of the specific binderswas verified by DNA sequencing.

Results:

The amino acid sequences of Fyn SH3 derived HER2 binders is presented inSEQ ID NOs: 167 to 318 as appended in the sequence listing.

Example 4.2 Fyn SH3 Derived Polypeptides Bind to Other Epitopes on HER2Compared to Anti-HER2 Antibodies

Methods:

The DNA sequences encoding FynSH3-derived clones C12 (SEQ ID NO: 167)and G10 (SEQ ID NO: 168) were subcloned into the bacterial expressionvector pQE12 so that the resulting constructs carried a C-terminalmyc-hexahistidine tag (SEQ ID NO: 328), and the two constructs wereexpressed and purified by means of the hexahistidine tag as described inGrabulovski et al. (Grabulovski et al. (2007) JBC, 282, p. 3196-3204).

The heavy and light chains (SEQ ID NO: 320 and SEQ ID NO: 321) of theanti-HER2 antibody 1 and the anti-HER2 antibody 2 (SEQ ID NO: 326 andSEQ ID NO: 329) were transiently co-expressed in CHO cells. Theantibodies were purified from the culture supernatant by affinitychromatography on a MabSelect SuRe column (GE healthcare).

10⁵ BT-474 cells (ATCC) were pre-incubated with an excess of 1 μManti-HER2 antibody 1, anti-HER2 antibody 2, or PBS for 60 min on ice.Subsequently, 300 nM C12 or G10 plus 20 nM mouse anti-myc antibody 9E10(Roche) were added to the cells without washing off the blockingantibodies. After 45 min incubation, cells were washed and boundC12/9E10- and G10/9E10 complexes were detected with anti-mouseIgG-Alexa488 conjugate. The cells were analyzed by FACS. Binding of C12and G10 to anti-HER2 antibody 1 or anti-HER2 antibody 2-blocked cellsurface was compared against binding to non-blocked cells. In order toanalyze the efficacy of the epitope blockade by anti-HER2 antibody 1 and2, 25 nM biotinylated antibody (biotinylation was performed withsulfo-NHS-LC-biotin (Pierce) according to the manufacturer'sinstructions) was added to the pre-blocked cells, followed by detectionwith Streptavidin-allophycocyanin conjugate.

Results:

The results of the experiments are shown in FIG. 27. Preblocking witheither of the antibodies drastically reduced binding of thecorresponding biotinylated antibodies, indicating that the preblockingstep efficiently and specifically blocked the epitopes of the twodifferent antibodies (FIG. 27B).

Binding of C12 and of G10 was not affected by preblocking with anti-HER2antibody 1 nor with anti-HER2 antibody 2, indicating that both clonesbind to an epitope different to anti-HER2 antibody 1 and anti-HER2antibody 2 (FIG. 27A).

Example 4.3 The Inventive Binding Molecules have a StrongerAntiproliferative Effect than the Combination of the Individual BindingProteins

HER2 targeting molecules with two different binding specificities werecreated by fusion of C12 via a glycine-serine (Gly₄Ser)₃ linker to theN-terminus of the light chain of anti-HER2 antibody 1 (resulting in theprotein termed COVA208) or anti-HER2 antibody 2 (termed COVA210).

Methods:

Anti-HER2 antibody 1 (SEQ ID NO: 320 and SEQ ID NO: 321), anti-HER2antibody 2 (SEQ ID NO: 326 and SEQ ID NO: 329), COVA208 (SEQ ID NO: 320and SEQ ID NO: 325) and COVA210 (SEQ ID NO: 326, SEQ ID NO: 327) weretransiently co-expressed in CHO cells and purified from the culturesupernatant by affinity chromatography on a MabSelect SuRe column (GEhealthcare). A bivalent monospecific format of clone C12 was created byfusion via a (Gly₄Ser)₃ to the C-terminus of human Fcγ1, resulting inFc-C12 (SEQ ID NO: 319). The protein was expressed and purified asdescribed above for anti-HER2 antibody 1, anti-HER2 antibody 2, COVA208and COVA210.

The growth inhibitory effect of the HER2 targeting constructs wasinvestigated in vitro on the NCl-N87 tumor cell line (purchased fromATCC). This human HER2 overexpressing gastric cell line was grown inRPMI1640 (Gibco) supplemented with 10% FBS (Gibco; heat inactivated at56° C. for 45 min). 7000 cells in 100 μl growth medium per well wereseeded into a 96-well plate. After incubation at 37° C./5% CO₂ for 24 h,20 μl of the anti-HER2 constructs Fc-C12, COVA208, COVA210, anti-HER2antibody 1 or anti-HER2 antibody 2, or combinations of the agents, wereadded. Each condition was performed in triplicate, and the agents wereadded in three-fold serial dilutions at concentrations between 300 nMand 0.015 nM. For combinations, each agent was used at the indicatedconcentration (e.g. 300 nM Fc-C12+300 nM anti-HER2 antibody 1). After 5days, the viability of the treated cultures was analyzed with XTT(Roche). The XTT reagent is converted by metabolically active cells intoa colored formazan product which absorbs light at 450 nm wavelength. Theabsorbance directly correlates with the live cell number. The %viability relative to PBS treated cells was calculated according to theformula:

${\%\mspace{14mu}{viablitiy}} = {( \frac{{OD}_{experimental} - {OD}_{blank}}{{OD}_{untreated} - {OD}_{blank}} ) \times 100}$

The average % viability was plotted against log₁₀(concentration), andthe resulting dose-response curves were analyzed by nonlinear regressionwith the software Prism, using the three parameter equation:

${\%\mspace{14mu}{viablitiy}} = {{bottom} + \frac{{top} - {bottom}}{1 + 10^{x - {{Lo}\;{gIC}_{50}}}}}$Results:

The fusion of Fyn SH3 derived binder C12 to the C-terminus of humanFcγ1, Fc-C12, did not have any effect on cell viability (FIGS. 28A and28C). When added in combination with anti-HER2 antibody 1 or anti-HER2antibody 2, Fc-C12 did not increase or decrease the activity of thesetwo antibodies significantly (FIGS. 28A and 28C). However, when cloneC12 was fused to the N-terminus of the light chain of the anti-HER2antibody 1 (COVA208) or anti-HER2 antibody 2 (COVA210) to generatemolecules with two different binding specificities for an antigen, itincreased the antiproliferative effect of the unmodified correspondingantibodies (FIGS. 28B and 28D).

In summary, these results show that the molecules COVA208 and COVA210are superior to the combination of the individual monospecific bindingproteins.

Example 4.4 The Anti-Proliferative Activity of Anti-HER2Fynomer-Antibody Fusions is Different Depending on the RelativeOrientation of the Fynomer and the Binding Site of the Antibody

Several different C12-antibody fusions were tested for their ability toinhibit growth of NCl-N87 tumor cells in order to investigate theinfluence of the fusion site where the Fyn SH3-derived sequence isattached to the antibody.

Methods:

COVA201 (SEQ ID NO:322; SEQ ID NO:321), COVA202 (SEQ ID NO:320; SEQ IDNO:323), COVA207 (SEQ ID NO:324; SEQ ID NO:321) and COVA208 (SEQ IDNO:320; SEQ ID NO:325) are all C12-anti-HER2 antibody 1 fusions in whichthe clone C12 is fused to either the C-terminus of the heavy chain(COVA201), C-terminus of the light chain (COVA202), N-terminus of theheavy chain (COVA207) and N-terminus of the light chain (COVA208).Expression and purification was performed as described for COVA208 inExample 4.3. The cell growth inhibition assay was performed on NCl-N87cells as described in Example 4.3.

Results:

The different C12-anti-HER2 antibody 1 formats were found to exhibitdifferent activities (ure 29A and 29B). COVA208 was most efficacious atinhibiting tumor cell growth and reduced the relative viability to 37%.COVA207 and COVA201 showed intermediate activity (viability: 52% and61%, respectively) while COVA202 was less active and reduced theviability to 67%, but was still better than anti-HER2 antibody 1 (81-82%viability).

These results show that fusions of one pair of a Fyn SH3-derivedsequence and an antibody have different activities, depending on thesite of fusion and that the N-terminal light chain fusion of C12 toanti-HER2 antibody 1 (═COVA208) showed the strongest anti-proliferativeefficacy.

Example 4.5 COVA208 Inhibits the Growth of BT-474 Cells with HigherEfficacy than Anti-HER2 Antibody 1

Methods:

The tumor cell growth inhibition of COVA208 (SEQ ID NOs: 320 and 325)was compared to anti-HER2 antibody 1 (SEQ ID NO: 320 and 321) on thehuman breast tumor cell line BT-474 (purchased from ATCC). This HER2overexpressing cell line is one of the best characterized models tostudy the activity of HER2 targeted agents. BT-474 cells were grown inDMEM/F12 medium (Gibco) supplemented with 10% heat-inactivated FBS(Gibco) and 10 μg/ml human recombinant insulin. The assay was performedas described in Example 4.3 for NCl-N87 cells.

Results:

COVA208 showed better antiproliferative activity than the anti-HER2antibody 1 (FIG. 30).

Example 4.6 COVA208 Inhibits NCl-N87 Tumor Growth In Vivo MoreEfficiently than the Anti-HER2 Antibody 1

COVA208 was investigated in vivo for tumor growth inhibition andcompared to anti-HER2 antibody 1.

Methods:

5×10⁶ human gastric tumor cells (ATCC; CRL-5822) were implanted s.c.into athymic CD-1 Nude mice (Charles River). Tumor dimensions and bodyweights were recorded three times weekly. The tumor volume wascalculated according to the formula volume=(width)²×length×π/6. When theaverage tumor size reached about 140 mm³, which was 42 days after tumorinoculation, mice were randomized into three treatment groups comprisingsix mice each, and the treatment was initiated. COVA208 (SEQ ID NOs: 320and 325) and anti-HER2 antibody 1 (SEQ ID NOs: 320 and 321) wereadministered i.p. once a week for four weeks (five injections in total).The first (loading) dose was 30 mg/kg, and each following (maintenance)dose was 15 mg/kg. Mice in the control group were injected with PBS.

Results:

Anti-HER2 antibody 1 treatment resulted in only weak tumor growthinhibition (FIG. 31). COVA208 showed improved tumor growth control forthe duration of the treatment compared to anti-HER2 antibody 1. On day32, the tumors in COVA208 treated mice were reduced in volume by 8%compared to the initial tumor size at the beginning of the treatment(d=0), whereas the anti-HER2 antibody 1-treated mice showed an increasein volume by 88%.

This result demonstrates that COVA208 shows significant superiorefficacy in vivo compared to anti-HER2 antibody 1.

Example 4.7 COVA208 Exhibits an Antibody-Like PK Profile In Vivo

Methods:

The pharmacokinetic profile of COVA208 in C57BL/6 mice (Charles River)was investigated and compared to anti-HER2 antibody 1. Three C57BL/6mice were injected i.v. with 200 μg COVA208 (SEQ ID NOs: 320 and 325) oranti-HER2 antibody 1 (SEQ ID NOs: 320 and 321). After 10 min, 6, 24, 48,96, 120, 144 and 168 hours, blood was collected into EDTA coatedmicrovettes (Sarstedt), centrifuged for 10 min at 9300 g and the serumlevels of COVA208 or anti-HER2 antibody 1 were determined by ELISA.Black maxisorp microtiter plates (Nunc) were coated with 50 nM HER2 ECD(Bender MedSystems). After blocking with 4% milk (Rapilait, Migros,Switzerland) in PBS, 40 μl of PBS and 10 μl of serum at appropriatedilution were applied. After incubation for 1 hr, wells were washed withPBS, and bound COVA208 or anti-HER2 antibody 1 were detected withprotein A-HRP conjugate (Sigma). The assay was developed with QuantaRedfluorogenic substrate (Pierce) and the fluorescence intensity wasmeasured after 5 to 10 min at 544 nm (excitation) and 590 nm (emission).The serum levels of COVA208 and anti-HER2 antibody 1 were determinedusing a standard curve of COVA208 and anti-HER2 antibody 1 (diluted to333-0.5 ng/ml each). From the concentrations of COVA208 and anti-HER2antibody 1 determined in serum at different time points and theresulting slope k of the elimination phase (plotted in asemi-logarithmic scale), the half-lives were calculated using to theformula t^(1/2)=ln 2/−k.

Results:

As shown in FIG. 32, the half-lives of COVA208 and the anti-HER2antibody 1 as determined from the elimination phase (beta phase,time-points 24 h-168 h) were highly similar (247 and 187 h,respectively). These data demonstrate that COVA208 has drug-like in vivoPK properties.

Example 4.8 COVA208 is Stable and does not Aggregate

The integrity and stability of COVA208 was assessed by SDS-PAGE and bysize exclusion chromatography.

Methods

Purified COVA208 (SEQ ID NOs: 320 and 325) and anti-HER2 antibody 1 (SEQID NOs: 320 and 321) were analyzed by SDS-PAGE. 4 μg protein were loadedeither with reduced or with nonreduced disulphide bonds onto a 4-12%Bis/Tris Novex gel in 1×MOPS running buffer (Invitrogen), together witha molecular weight marker (RPN800e; GE healthcare). Protein bands werevisualized by coomassie staining.

The size exclusion chromatography (SEC) profile of COVA208 wasdetermined immediately after purification as well as after storage ofthe protein in PBS at 4° C. for one or two months. 100 μl COVA208 at aconcentration of 1.75 mg/mL was loaded onto a Superdex 200 10/300 GLcolumn in PBS (GE healthcare) at a flow rate of 0.5 ml/min, and theelution from the column was monitored by reading the OD₂₈₀.

Results:

The results of the SDS-PAGE and the SEC profiles of COVA208 are shown inFIG. 33. COVA208 runs in clearly defined bands at the expected molecularweight on an SDS-PAGE (top). Of particular interest is the finding thatthere is no native light chain detectable in COVA208 (MW around 30 kDa),indicating that there is no cleavage of the Fyn SH3-derived clone C12from the antibody light chain.

COVA208 eluted in one main peak form the SEC column with a retentionvolume of 13.1 ml (bottom). Anti-HER2 antibody 1 eluted at 13.2 ml. Mostimportantly, no aggregates, which would elute at around 8 ml, weredetectable in the COVA208 protein preparation. The SEC profile ofCOVA208 did not change over two months of storage at 4° C. The elutionpeak remained narrow, symmetrical and appeared at the same retentionvolume. The protein preparation remained free of aggregates after 1 and2 months of storage. This indicates that COVA208 remains stable overextended periods of storage at 4° C. In summary, these results supportthat COVA208 is a stable, monodisperse molecule with optimal biophysicalproperties.

Example 4.9 COVA208 has Superior Growth Inhibitory Activity as Comparedto Anti-HER2 Antibody 1 on a Panel of Ten HER2-Expressing Tumor CellLines

The anti-proliferative activity of COVA208 (SEQ ID NOs: 320 and 325) wascompared to anti-HER2 antibody 1 (SEQ ID NOs: 320 and 321) on differentHER2 positive cell lines. XTT assays were performed essentially asdescribed in example 4.3. The cell lines used in this experiment and theexperimental conditions are given in Table 12. Dose-response curves werefitted to the three parameter equation as described in example 4.3, andthe maximal growth inhibition was calculated with the formula:Maximum level of inhibition (%)=100%−bottom

With the variable bottom derived from the nonlinear regression of thedose-response curves using the formula:

${\%\mspace{14mu}{viablitiy}} = {{bottom} + \frac{{top} - {bottom}}{1 + 10^{x - {{Lo}\;{gIC}_{50}}}}}$

The results of these assays are shown in FIG. 34. FIGS. 34A and 34B showdose-response curves obtained on the OE19 and on the Calu-3 cell lines,respectively. FIG. 34C represents the maximal growth inhibition obtainedon each cell line with COVA208 and anti-HER2 antibody 1, including theresults on NCl-N87 and BT-474 cell lines shown in FIGS. 28 and 30.COVA208 shows improved anti-proliferative activity as compared toanti-HER2 antibody 1 on all 10 cell lines.

Example 4.10 COVA208 Induces Apoptosis in NCl-N87 Gastric Cancer Cells

The ability of COVA208 to induce apoptosis was investigated on NCl-N87cells by analyzing caspase 3/7 enzymatic activity and by detecting DNAfragmentation by TUNEL staining.

Methods

Caspase 3/7 assay: 45′000 NCl-N87 cells were seeded into the wells of a96-well microtiter plate. One day later, 100 nM anti-HER2 antibody 1(SEQ ID NOs: 320 and 321), COVA208 (SEQ ID NOs: 320 and 325) or PBS wereadded to the cells in triplicate. As positive control, 1 μMstaurosporine was added. After two days incubation, the activity ofcaspase-3 and caspase-7 was determined using the fluorescence Apo-ONE®homogenous caspase-3/7 kit (Pierce).

The viability of the treated cultures was analyzed by XTT in parallel onreplica plates, and the % viability relative to PBS treated samples wascalculated as described in example 4.3.

Caspase 3/7 activity was divided by % viability to obtain the normalizedcaspase 3/7 activity.

TUNEL assay: 0.8×10⁶ NCl-N87 cells in 2 mL were distributed in 6-wellplates. On the next day, 300 nM anti-HER2 antibody 1 (SEQ ID NOs: 320and 321), COVA208 (SEQ ID NOs: 320 and 325) or PBS were added to thecells. As positive control, 1 μM staurosporine was added. After threedays incubation, cells were detached, formalin-fixed, permeabilized in70% ice-cold ethanol and the 3′-hydroxyl DNA ends labeled withfluorescein-deoxyuridine triphosphate (FITC-dUTP), using the APO-DIRECTkit (Phoenix flow systems). Labeled cells were analyzed by FACS, and the% TUNEL-positive cells determined by gating on the FITC-dUTP positivecell population.

Results

The results of the caspase 3/7 assay are shown in FIG. 35A. COVA208resulted in increased caspase 3/7 activity, indicating that COVA208induced apoptosis in NCl-N87 cells. Anti-HER2 antibody 1 did not resultin induced caspase 3/7 activity.

The results of the TUNEL assay are shown in FIG. 35B. COVA208 inducesDNA fragmentation in the majority of cells, further supporting that itis capable of inducing apoptosis, whereas anti-HER2 antibody 1 is not.

Example 4.11 COVA208 Inhibits Ligand-Dependent and Ligand-IndependentHER2-Mediated Signalling

Activation of HER2 downstream signaling leads to phosphorylation ofHER3, resulting in the activation of the PI3K-Akt-mTOR pathway, or tothe activation of the MAPK/Erk pathway. In tumor cell lines that displaysufficiently high surface density of HER2, these downstream pathways areconstitutively activated in the absence of HER3 ligands(ligand-independent signaling). In addition to ligand-independentactivation of HER2 downstream signalling, the downstream pathways canalso be activated by HER3 ligands which promote HER2-HER3 heterodimerformation (ligand-dependent signaling).

In order to investigate the effects of COVA208 on HER2 downstreamsignaling, HER2-overexpressing NCl-N87 cells were treated with COVA208(SEQ ID NOs: 320 and 325), anti-HER2 antibody 1 (SEQ ID NOs: 320 and321), anti-HER2 antibody 2 (SEQ ID NOs: 326 and 329), or PBS, and thecell lysates were analyzed for phospho-proteins by immunoblotting.

The assay was also performed on HER2 low-expressing MCF-7 cells, inwhich HER2 downstream phosphorylation is triggered only after additionof the HER3 ligand heregulin-1β.

Methods

NCl-N87 cells (ATCC; CRL-5822) were distributed in 6-well culture dishesin complete medium at 1×10⁶ cells in 3 mL per well. After overnightincubation at 37° C./5% CO₂, 40 μg/mL anti-HER2 agents were added andthe cells were incubated at 37° C./5% CO₂ for 72 h. Cells weresubsequently lysed on ice in cell lysis buffer containing 1% Triton-X,protease inhibitor and phosphatase inhibitor cocktails (Roche AppliedSciences).

MCF-7 cells (ATCC; HTB-22) were cultured in MEM (Gibco)+10% FBS (Gibco).Cells were distributed in 6-well culture dishes at 0.5×10⁶ cells in 3 mLper well. After overnight incubation at 37° C./5% CO₂, cells werestarved in medium without serum for 3 h. 40 μg/mL anti-HER2 agents werethen added for 1 h during which the cells were kept at 37° C./5% CO₂.After 45 min, 2 nM human recombinant heregulin-1β (R&D systems) wasadded for 15 min. Cells were subsequently lysed on ice in cell lysisbuffer containing 1% Triton-X, protease inhibitor and phosphataseinhibitor cocktails (Roche Applied Sciences).

Total cell lysates were cleared by centrifugation at 16′000×g for 10 minat 4° C. and the protein concentration in the cleared lysates wasdetermined by Bradford assay (Bio-Rad). 10 μg of protein were separatedon Novex® 4-12% Bis-Tris gels (Invitrogen) and transferred onto PVDFmembrane.

Phospho-proteins were detected on PVDF membrane with antibodies againstpHER3^(Y1289) (Millipore), pAkt^(S473) (CST) or pErk1/2^(T202/Y204)(CST), followed by secondary HRP-conjugated antibodies (Jackson ImmunoResearch). Vinculin was detected with a vinculin-specific antibody(Millipore) and served as loading control. The immunoblots weredeveloped with ECD prime chemiluminescent HRP substrate (GE healthcare)and exposed onto X-Ray film.

Results:

The results of this experiment are shown in FIG. 36. In MCF-7 cells, inwhich activation of HER2 downstream signaling requires HER3 ligands,COVA208 and anti-HER2 antibody 1 both block phosphorylation of HER3, Aktand Erk1/2 equally well, indicating that COVA208 retained the activityof its parental antibody. In contrast, anti-HER2 antibody 2 does notblock ligand-induced phosphorylation of HER3, Akt or Erk1/2.

In NCl-N87 cells, where phosphorylation of HER2 downstream signalingproteins occurs independent of HER3 ligands, COVA208 efficiently blocksphosphorylation of HER3, Akt or Erk1/2, whereas anti-HER2 antibody 1does not block phosphorylation. Anti-HER2 antibody 2 is also capable ofefficiently blocking HER2 signaling under these conditions. Theseresults indicate that COVA208 blocks ligand-dependent as well asligand-independent HER2 downstream signalling events, in contrast toanti-HER2 antibodies 1 and 2, which block one but not the other.

Example 4.12 COVA208 is Internalized by NCl-N87 Cells

In order to investigate whether COVA208 promotes internalization of theHER2 receptor in vitro, NCl-N87 cells were cultured in the presence ofCOVA208 (SEQ ID NOs: 320 and 325) or with anti-HER2 antibody 1 (SEQ IDNOs: 320 and 321) followed by fixation and permeabilization of the cellsand subsequent detection of the anti-HER2 agents by means of afluorescent secondary antibody. Microscopic imaging was used to assessthe sub-cellular distribution of the fluorescent signal.

Methods

NCl-N87 cells grown in Lab-Tek II CC² chamber slide wells were surfacelabelled on ice for 1 h with 100 nM COVA208 or anti-HER2 antibody 1.Unbound anti-HER2 agent was then washed off. As positive control, 1 μMgeldanamycin (Hsp90 inhibitor) which causes rapid internalization ofHER2 was added to some wells. The cells were transferred to 37° C./5%CO₂ for 0 h or 5 h to allow for internalization, then fixed withformalin and permeabilized with saponin. An Alexa488-labeled anti-humanIgG antibody (Invitrogen) was used to detect anti-HER2 agents onpermeabilized cells, and nuclei were stained with Hoechst 33342 dye. Thestained cells were analyzed on a Leica TCS SP2-AOBS laser scanningconfocal microscope. Optical sections (z-stacks, d=0.2 μm) werecollected and three regions were analyzed. The amount of anti-HER2agents which localized into distinct dots was quantified with thesoftware Imaris 7.4.0 (Bitplane), using the surface tool of Imaris todetected spheroid dots, and expressing the percentage of anti-HER2agents present in dots:% anti-HER2 agents in dots=(volume of dots/volume of total anti-HER2staining)×100Results

After surface labelling and before incubation at 37° C., COVA208 andanti-HER2 antibody 1 localized to the cell membrane. After 5 hoursincubation at 37° C., COVA208 was present in distinct dots within thecytosol, while the cell membrane was only very weakly stained. Incontrast, the anti-HER2 antibody 1 was confined to the cell membraneafter 5 h incubation at 37° C., and only very few dots in the cytosolwere detected. If co-incubated with geldanamycin, anti-HER2 antibody 1was also found in dots and the cell membrane was negative for theantibody. These results indicate that unlike anti-HER2 antibody 1,COVA208 rapidly internalizes into NCl-N87 cells.

The quantification of the % staining appearing within dots is shown inFIG. 37. The majority of COVA208 localizes into dots, whereas only asmall fraction of anti-HER2 antibody 1 is found in dots.

Example 4.13 COVA208 Inhibits KPL-4 Breast Tumor Growth In Vivo MoreEfficiently than the Anti-HER2 Antibody 1

COVA208 was investigated in vivo in KPL-4 breast tumors for growthinhibition and compared to anti-HER2 antibody 1.

Methods:

3×10⁶ human KPL-4 breast tumor cells (Kurebayashi et al. (1999) Br. J.Cancer. 79; 707-717) were implanted into the mammary fat pad of femaleSCID beige mice (Charles River). Tumor dimensions and body weights wererecorded three times weekly. The tumor volume was calculated accordingto the formula volume=(width)²×length×n16. When the average tumor sizereached 70 mm³, mice were randomized into three treatment groupscomprising eight mice each, and the treatment was initiated. COVA208(SEQ ID NOs: 320 and 325), anti-HER2 antibody 1 (SEQ ID NOs: 320 and321) or PBS were administered i.p. once a week for four weeks (fiveinjections in total). The first (loading) dose was 30 mg/kg, and eachfollowing (maintenance) dose was 15 mg/kg.

Results:

Anti-HER2 antibody 1 treatment resulted in very weak tumor growthinhibition only (FIG. 38). COVA208 showed significantly improved tumorgrowth control. This result further supports that COVA208 showssignificantly superior efficacy in vivo compared to anti-HER2 antibody1.

Example 4.14 Determination of the HER2 Epitope Bound by the FynSH3-Derived Polypeptide C12

The epitope bound by the Fyn SH3-derived clone C12 (SEQ ID NO: 167) onHER2 was identified by an alanine scanning mutation approach and wasperformed at Integral Molecular Inc. (Philadelphia, USA). A shotgunmutagenesis mutation library was created as described in Paes et al(2009) J Am Chem Soc 131(20): 6952-6954. Briefly, a eukaryoticexpression plasmid encoding full-length human HER2 was constructed witha C-terminal V5H is epitope tag. Using the parental cDNA construct as atemplate, alanine scanning mutations were introduced into theextracellular domain of HER2 (amino acids 23-652 of SEQ ID NO: 337)using PCR-based mutagenesis. Residues which were already alanine in theparental construct were mutated to methionine. Mutated constructs andthe parental HER2 control construct were expressed in HEK-293T cells.Twenty-four hours post-transfection, cells were washed in PBS and fixedin 4% paraformaldehyde. Cells were incubated with control anti-HER2monoclonal antibody (MAB1129, R&D Systems) or with Fyn SH3-derived cloneC12 (expressed as N-terminal Fc fusion) in PBS with Ca^(2±)/Mg²⁺ (PBS++)and 10% Normal Goat Serum (NGS) for 1 hour. After two washes in PBS,cells were incubated with goat anti-human Alexa Fluor 488-conjugatedsecondary antibodies (Jackson, West Grove, Pa.) in PBS++ and NGS for 1hour, followed by 2 washes in PBS. Microplates were measured by flowcytometry using the Intellicyt HTFC Screening System and quantifiedusing Forecyt software (Intellicyt Corporation, Albuquerque, N. Mex.).

It has been found that the Fyn SH3-derived polypeptide C12 (SEQ ID NO:167) binds to an epitope of HER2 which is located within domain I ofHER2 (SEQ ID NO: 338). In more detail, five alanine scanning mutationswere identified which resulted in markedly reduced binding of thebinding molecules comprising the Fyn SH3-derived polypeptide C12 (SEQ IDNO: 167) while binding of the control antibody MAB1129 was retained.These mutations included T166A, R188A, P197A, S202A and R203A ascompared to the sequence of SEQ ID NO: 338. In other terms, at leastamino acid positions T166, R188, P197, S202 and R203 of domain I of HER2are involved in binding between the Fyn SH3-derived polypeptide C12 andHER2.

TABLE 12 HER2 expressing cell lines used in in vitro proliferationassays described in XTT assay conditions incuation time with cells/wellanti-HER2 Cell line Description Distributor growth medium seeded agentsNCI-N87 gastric carcinoma, liver metastasis ATCC RPMI1640 + 10% FBS 70005 days BT-474 breast, ductal carcinoma ATCC DMEM/F12 + insulin + 7000 5days 10% FBS KPL-4 breast, malignant pleural effusion Prof.Kurebayashi * DMEM + 10% FBS 2000 3 days OE19 gastric (oesophagalcarcinoma) hpa cultures RPMI1640 + 10% FBS 5000 5 days Calu-3 pleuraleffusion of lung adenocarcinoma ATCC MEM + 10% FBS 5000 5 days SKOV-3ovarian adenocarcinoma, ascites ATCC modified McCoy5a + 2000 3 days 10%FBS MDA-MB-453 pericardial effusion of metastatic breast carc. ATCCDMEM + 10% FBS 2000 5 days HCC202 primary ductal carcinoma ATCCRPMI1640 + 10% FBS 5000 5 days ZR-75-30 breast, ductal carcinoma,malignant ascites ATCC RPMI1640 + 10% FBS 5000 5 days MDA-MB-175-VIIpleural effusion of ductal carcinoma ATCC DMEM + 10% FBS 5000 5 days *Kurebayashi et al. (1999) Br. J. Cancer. 79; 707-717

FIG. 34 and the conditions applied in the in vitro proliferation assays.

Example 5 Anti-Human Serum Albumin Fyn SH3 Derivatives Example 5.1 FynSH3 Derived Polypeptides Bind to Human Serum Albumin

Methods

1) Lysate ELISA on Human Serum Albumin Protein

Using the Fynomer® phage libraries described in Schlatter et al.(Schlatter et al. (2012) mAbs, 4(4) p. 497-50) Fyn-SH3 derived bindingproteins specific to human serum albumin were isolated using human serumalbumin (Sigma-Aldrich, cat. no A3782) and serum albumin from a rodentspecies (rat serum albumin, Sigma-Aldrich, cat. no A6414) as antigensand standard phage display as selection technology (Grabulovski D. etal., (2007) J Biol Chem 282, p. 3196-3204, Viti, F. et al. (2000)Methods Enzymol. 326, 480-505).

After naïve and affinity maturation selections, enriched Fyn SH3-derivedpolypeptides were screened for binding to human serum albumin and/orserum albumin from a rodent species (mouse/rat) by lysate ELISA. DNAencoding the Fyn SH3-derived binding proteins was cloned into thebacterial expression vector pQE12 (Qiagen) so that the resultingconstructs carried a C-terminal myc-hexahistidine tag as described inGrabulovski et al. (Grabulovski et al. (2007) JBC, 282, p. 3196-3204).The polypeptides were expressed in the cytosol of E. coli bacteria in a96-well format and 200 ml of cleared lysate per well was preparedessentially as described in Bertschinger et al. (Bertschinger et al.(2007) Protein Eng Des Sel 20(2): p. 57-68). Briefly, transformedbacterial colonies were picked from agar plates and grown in a roundbottom 96-well plate (Nunc, cat. no. 163320) in 200 μl 2×YT mediumcontaining 100 μg/ml ampicillin and 0.1% (w/v) glucose. Proteinexpression was induced after growth for 3 h at 37° C. and 200 r.p.m. byadding 1 mM IPTG (Applichem, Germany). Proteins were expressed overnightin a rotary shaker (200 r.p.m., 30° C.). Subsequently, the 96-well platewas centrifuged at 1800 g for 10 min and the supernatant was discarded.The bacterial pellets were resuspended in 65 μl Bugbuster containingBenzonase Nuclease (VWR, cat. No. 70750-3) and incubated at RT for 30minutes. Afterwards, the monoclonal bacterial lysates were cleared bycentrifugation (1800 g for 10 min), diluted with 170 μL PBS and filteredusing a multiscreen filter plate (0.45 μm pore size; Millipore cat. No.MSHVN4510). Monoclonal bacterial lysates were used for ELISA: humanserum albumin was immobilized on maxisorp F96 wells (Nunc, cat. no439454) overnight at room temperature. Plates were then blocked withPBS, 4% (w/v) milk (Rapilait, Migros, Switzerland). Subsequently, 20 μlof PBS, 10% milk containing 25 μg/ml anti-myc antibody 9E10 and 80 μl ofbacterial lysate were applied (resulting in a final anti-myc antibodyconcentration of 5 mg/ml). After incubating for 1 h and washing, boundFyn SH3-derived polypeptides were detected with anti-mouse-HRP antibodyconjugate (Sigma) at a final concentration of 5 μg/ml. The detection ofperoxidase activity was done by adding 100 μL per well BM blue PODsubstrate (Roche) and the reaction was stopped by adding 50 μl 1 MH₂SO₄. The DNA sequence of the specific binders was verified by DNAsequencing. Cross-reactivity towards serum albumin from a rodent specieswas detected by monoclonal lysate ELISA using mouse serum albumin(Sigma-Aldrich, cat. no A3139) as an antigen and the protocol describedabove. Alternatively, cross-reactivity towards mouse and rat serumalbumin was confirmed surface plasmon resonance experiments (see below).

2) Expression and Purification of Fyn SH3-Derived Polypeptides in E.coli

Fyn SH3-derived albumin-binding polypeptides were expressed in thecytosol of TG1 E. coli bacteria as well as purified as described inGrabulovski et al. (Grabulovski et al. (2007) JBC, 282, p. 3196-3204).

3) Affinity Measurements

Affinity measurements were performed using a Biacore T200 instrument (GEHealthcare). For the interaction analysis between serum albumin, derivedfrom mouse, rat or human, and Fyn SH3-derived albumin-bindingpolypeptides, a Series S CM5 chip (GE Healthcare) was used with albuminproteins immobilized using the amine coupling kit (GE healthcare). Serumalbumin proteins from different species (mouse, rat or human) wereimmobilized (2000-3000 RU) on different flow cells of the chip whereas ablank-immobilized flow cell served as a reference flow cell. The runningbuffer was PBS containing 0.05% Tween 20 at pH 7.4. The interactionswere measured at a flow of 30 μl/min and 25° C. and differentconcentrations of Fyn SH3-derived albumin-binding polypeptides wereinjected. All kinetic data of the interaction was evaluated usingBiacore T200 evaluation software.

Results

1) The amino acid sequences of ELISA positive Fyn SH3-derivedpolypeptides binding to human serum albumin is presented in SEQ ID NOs:340 to 376 as appended in the sequence listing. In addition, FynSH3-derived polypeptides (SEQ ID NOs: 340 to 368) also showed binding tomouse serum albumin as confirmed by lysate ELISA and/or Biacore affinitymeasurements.2) The expression yields of two selected Fyn SH3-derived albumin-bindingpolypeptides of the invention from bacterial cultures undernon-optimized conditions in shake flasks is depicted in Table 13. Theyield was in the same range as the expression yield of the WT Fyn-SH3polypeptide. High protein-purity was confirmed by SDS-PAGE analysis andthe gel is depicted in FIG. 39.

TABLE 13 Expression yields of Fyn SH3-derived albumin-bindingpolypeptides produced in TG1 E.coli bacteria Fynomer ® SEQ ID NO. Yield(mg/l) 17H 341 10 C1 340 25 WT Fyn-SH3 339 103) The binding properties were analyzed by real-time interactionanalysis on a Biacore chip revealing the following dissociationconstants (K_(D)) for selected albumin-binding polypeptides againstalbumin derived from either rat (RSA), mouse (MSA) or human (HSA)(depicted in Table 14).

TABLE 14 Dissociation constants of Fyn SH3-derived serum albumin-bindingpolypeptides to RSA, MSA and HSA. SEQ ID K_(D) (nM) K_(D) (nM) K_(D)(nM) Fynomer ® NO. RSA MSA HSA C1 340 72 408 1290 17H 341 17 96 455

Example 5.2 Albumin-Binding Fyn SH3 Derived Polypeptides have aProlonged Serum Half-Life in Mice

Methods

The pharmacokinetic profile of albumin-binding Fyn-SH3 derivedpolypeptides was investigated in BALB/c mice (Charles River) andcompared to the WT Fyn-SH3 molecule. Fynomer® C1 (SEQ ID NO: 340),Fynomer® 17H (SEQ ID NO: 341) and WT Fyn-SH3 (SEQ ID NO: 339) wereradiolabeled using Iodine-125 (Perkin Elmer cat no. NEZ033A001MC) andChloramine T (Sigma-Aldrich cat NO 31224). The labeling reaction wascarried out for two minutes at room temperature before removal oflabeling reagents using PD MiniTrap G-25 columns (GE Healthcare cat. no28-9180-07). Three BALB/c mice were injected i.v. with 13.5 μg of eitherradiolabeled Fynomer® C1 (SEQ ID NO: 340), Fynomer® 17H (SEQ ID NO: 341)or WT Fyn-SH3 (SEQ ID NO: 339). After 10 minutes, 2.5, 4, 6, 9, 25, 35hours, blood was collected into EDTA coated microvettes (Sarstedt) andcentrifuged for 10 min at 9300 g. Radioactivity was counted by mixingthe serum with Supermix Perkin Elmer Scintillation Fluid andquantification of beta-emission of each sample with a 1450 MicroBetaTrilux scintillation counter and serum levels were calculated (resultsexpressed as % injected dose (ID)/ml of blood). From the serum levels ofFynomer® C1, Fynomer® 17H and WT Fyn-SH3 determined in serum atdifferent time points and the resulting slope k of the elimination phase(plotted in a semi-logarithmic scale), the half-lives were calculatedusing the formula t_(1/2)=ln 2/−k.

Results

As depicted in Table 15, Fynomer® C1 (SEQ ID NO: 340) and Fynomer® 17H(SEQ ID NO: 341) show a significantly better terminal half-life as theWT Fyn-SH3 protein (SEQ ID NO: 339). Time-points used for half-lifecalculation: Fynomer® C1 and Fynomer® 17H: 2.5-35 h; WT Fyn-SH3: 2.5-25h)

TABLE 15 Terminal half-life of Fyn SH3-derived serum albumin-bindingpolypeptides in mice compared to the WT Fyn-SH3 protein. SEQ IDFynomer ® NO: t_(1/2) (h) C1 340 10.5 17H 341 21.3 WT Fyn-SH3 339 4.4

Example 5.3 Albumin-Binding Fyn SH3 Derived Polypeptides can ExtendSerum Half-Life of BITE® Molecules

Methods:

1) Expression and Purification of an Albumin Binding Fyn-SH3 FusionProtein

The Fynomer® 17H (Seq ID NO: 341) has been genetically fused to theN-terminus of the CD3-PSMA specific BITE® (Seq ID NO: 378) via a 15amino acid linker (linker SEQ ID NO: 377) yielding the trispecificanti-albumin/PSMA/CD3 protein COVA406 (SEQ ID NO: 379). The BITE®protein (SEQ ID NO: 378) and the fusion molecule of the inventionCOVA406 (SEQ ID NO: 379) carrying a C-terminal penta-his-tag weretransiently transfected into FreeStyle CHO-S cells and expressed inserum-free/animal component-free media for 6 days. The proteins werepurified from the supernatants by Protein L affinity chromatography(Thermo Scientific, cat. No. 89928) with an ÄKTA Purifier instrument (GEHealthcare). Concentrations were determined by absorbance measurement at280 nm. Yields are listed in Table 16. The SDS PAGE of both proteins isshown in FIG. 40.

After purification size exclusion chromatography has been performed withCOVA406 using an ÄKTA FPLC system and a Superdex G200, 30/100 GL column(GE Healthcare) (see FIG. 41).

2) FACS Binding Experiment with a BITE® Fusion Protein of the Invention

The polypeptide COVA406 (SEQ ID NO: 379, final concentration 300 nM) wasmixed with 100 μl cell suspension containing either (i) 1×10⁵ JurkatE6-1 cells (CD3 positive cells), (ii) 1×10⁵ 22Rv1 prostate carcinomacells (PSMA positive cells) or (iii) 1×10^(5 LS)174T colorectaladenocarinoma cells (PSMA and CD3 negative, ATCC cat. No. CL-188) inPBS/1% BSA/0.2% sodium azide. As a negative control, the same cells wereincubated with PBS/1% BSA/0.2% sodium azide instead of COVA406 (PBScontrol). After 60 min incubation on ice, cells were washed, and boundprotein was detected by incubation with 10 μg/ml mouse anti tetra-HISantibody (Qiagen, cat no. 34670), followed by incubation with anti-mouseIgG-Alexa488 conjugate (Invitrogen) at a concentration of 10 ug/mL.Finally cells were washed three times and stained cells were thenanalyzed on a Guava easyCyte™ (Millipore) flow cytometer.

3) Redirected T-Cell Mediated Cell Cytotoxicity Analysis

The polypeptide COVA406 (SEQ ID NO: 379) was tested in a redirectedT-cell mediated cell cytotoxicity assay using a protocol adapted fromDreier et. al. (2002) Int. J. Cancer: 100, 690-697.

Human PBMCs were used as effector cells. On the day before theexperiment PBMCs were isolated from fresh buffy coat preparations byFicoll Plaque plus (GE Healthcare) and density gradient centrifugationusing standard procedures. Isolated PBMCs were then incubated over nightat a cell concentration of 4×10⁶ cells/ml in 10% FCS, RPMI and 37° C.,5% CO₂.

For the cell kill experiment PBMCs were centrifuged and resuspended in10% FCS, RPMI at a cell concentration of 2.5×10⁷ cells/ml.

Target cells were labeled with Calcein AM by incubating cells at a finalCalcein AM concentration of 10 μM for 30 min at 37° C., 5% CO₂.Subsequently excess dye was removed by washing cells twice with approx.15 mL Medium. Finally target cell number was adjusted to 1*10⁶ cells/ml.Target tumor cells were either 22Rv1 cells (PSMA positive, ATCC cat. No.CRL-2505) or HT29 colon carcinoma cells (PSMA negative, DSMZ cat. No.ACC-299).

Effector molecules were diluted in 10% FCS, RPMI to a maximumconcentration of 1200 ng/mL. A dilution series of 1/10 dilutions wasprepared.

Finally target cell suspension, effector cell suspension and thedifferent concentrations of the polypeptide COVA406 (SEQ ID NO: 379)were then mixed in equal amounts. A total of 50000 target cells wereadded per well and the effector to target cell ratio was 25/1, The finalmaximal concentration of effector molecules was 400 ng/μl. Cell lysiswas measured after 5 hours incubation at 37° C. and 5% CO₂. Afterincubation, the cell suspension was centrifuged and cell lysis wasquantified by detection of Calcein AM fluorescence in the supernatantusing a fluorescence reader.

The amount of redirected cell lysis was normalized to the maximum lysiscontrol (cells lysed by the addition of 1% Triton X-100) and spontaneouslysis (target cells incubated with PMBCs in the absence of effectormolecules). Percentage of cell lysis was calculated according to thefollowing formula:% lysis=(((fluorescence sample)−(fluorescence spontaneous lysiscontrol))/((fluorescence maximum lysis control)−(fluorescencespontaneous lysis control)))×100

All measurements were done in triplicates. Specific cell lysis wasplotted versus the concentration of COVA406 and evaluated using Prism 5(GraphPad Software) by fitting a sigmoidal dose-response.

4) Comparison of the Pharmacokinetic Profiles of COVA406 and the BITE®Molecule

The pharmacokinetic profile of COVA406 in C57BL/6 mice (Charles River)was investigated and compared to the parental BITE® molecule. FiveC57BL/6 mice were injected i.v. each with 48 μg COVA406 (SEQ ID NO: 379)or BITE® (SEQ ID NO: 378). After 10 and 30 min, 1, 3, 5, 7, 9, 12, 24,28, 33 and 48 hours, blood was collected into EDTA coated microvettes(Sarstedt), centrifuged for 10 min at 9300 g and the serum levels ofCOVA406 or BITE® were determined by ELISA. Briefly, black maxisorpmicrotiter plates (Nunc) were coated with 10 μg/ml of a peptide derivedfrom CD3 (Sequence: QDGNEEMGGITQTPYKVSISGTTVILT; SEQ ID NO: 380)(expressed as Fc-fusion) and incubated over night at 4° C. Afterblocking with 4% milk (Rapilait, Migros, Switzerland) in PBS, serumsamples at appropriate dilutions were applied, resulting in a finalbuffer concentration of 2% mouse serum (Sigma) and 4% milk. Afterincubation for 1 hr, wells were washed with PBS, and bound COVA406 orBITE® were detected with Penta-His-biotin (Qiagen) followed byStreptavidin-HRP conjugate (Sigma). The assay was developed withQuantaRed fluorogenic substrate (Pierce). The reaction was stopped after3 min incubation and the fluorescence intensity was measured at 544 nm(excitation) and 590 nm (emission). The serum levels of COVA406 andBITE® were determined using a standard curve of COVA406 and BITE®(diluted to 333-0.5 ng/ml each). From the concentrations of COVA406 andBITE® determined in serum at different time points and the resultingslope k of the elimination phase (plotted in a semi-logarithmic scale),the half-lives were calculated using the formula t_(1/2)=ln 2/−k.Timepoints used for half-life calculation: COVA406: 1-48 h; BITE®: 1-12h.

Result:

COVA406 (SEQ ID NO: 379) expressed with a similar yield as the BITE®molecule (SEQ ID NO: 378) (Table 16).

TABLE 16 Purification yields of the BITE ® and Fyn-SH3 derivedalbumin-binding polypeptide fusions produced in transiently transfectedCHO-S cells. SEQ ID NO: Yield (mg/l) BITE ® 378 8.1 COVA406 379 5.0

The size exclusion chromatography (SEC) profile after purificationdemonstrated that COVA406 eluted as a single, monomeric peak showingthat the fusion protein has excellent biophysical properties (FIG. 41).Specific binding to PSMA-positive cells (22Rv1 cells) and CD3-positivecells (Jurkat E6-1, CD3 positive) was validated in a FACS experiment.Mean fluorescence intensities (MFI) of the stainings are depicted inFIG. 42. Redirected T-cell mediated cell cytotoxicity was validated in aCalcein release assay using PBMCs as effector cells. Specific redirectedcell-lysis of PSMA-positive cells with COVA406 (EC₅₀=4.35 ng/ml) isshown in FIG. 43. Cells with no PSMA expression (HT29 cells) were notlysed under the same conditions, showing that COVA406 is able to killspecifically PSMA positive cells. An improved pharmacokinetic profile ofCOVA406 (SEQ ID NO: 380) compared to the BITE® protein (SEQ ID NO: 379)was observed in mice. FIG. 44 shows the serum concentrations (ng/ml) andterminal elimination phase of COVA406 and the parental BITE®. COVA406shows a significantly better half-life (14.3 hours) compared to theBITE® (1.5 hours). This example shows that serum albumin bindingproteins of the invention are able to prolong the in vivo half-life ofotherwise short-lived molecules, in particular of BITE® molecules.

Example 5.4 Prior Art Fynomers® which Bind to Serum Albumin

For Material and Methods, see Publications EP2054432 and “Grabulovski,Dragan: The SH3 domain of fyn kinase as a scaffold for the generation ofnew binding proteins. ETH Dissertation Nr 17216 (May 2007).http://dx.doi.org/10.3929/ethz-a-005407897”.

FIG. 45 shows specificity ELISA of Fyn SH3 variants isolated afteraffinity selections. None of the Fynomers® binds to HSA or HSA/rodentserum albumin, except for C3. However, C3 cross-reacts also with thenon-related ovalbumin (hen egg white albumin). Therefore, C3 isconsidered as an unspecific binding protein.

Example 6 Anti-Human Anti-her2/EGFR/CD33 Fyn SH3 Derivatives Example 6.1Redirected T-Cell Mediated Cell Cytotoxicity Analysis Towards HER2Positive Tumor Cells Example 6.1.1 Expression and Purification ofAnti-CD3 Antibody Anti-HER2 Fynomer® Fusion Proteins

The HER2 binding Fynomer® C12 (Seq ID NO: 383) has been geneticallyfused to the anti CD3 binding antibody (SEQ ID NOs: 381 and 382) via a15 amino acid linker (SEQ ID NO: 386) yielding the bispecific antibodyFynomer® fusion proteins of the present invention. In COVA420 theFynomer® C12 was fused to the N-terminus of the heavy chain of the antiCD3 antibody (SEQ ID NOs: 381 and 382). In COVA422 the Fynomer® C12 wasfused to the C-terminus of the heavy chain of the anti CD3 antibody (SEQID NO: 381 and 382).

The anti-CD3 antibody (SEQ ID NO: 381 and 382), COVA420 (SEQ ID NO: 387and 388), COVA422 (SEQ ID NO: 389 and 390) and the anti-CD3×anti-HER2scFv control (SEQ ID NO: 391) (carrying a hexa-his tag), weretransiently transfected into FreeStyle CHO-S cells and expressed inserum-free/animal component-free media for 6 days. The anti-CD3 antibodyand the bispecific proteins of the invention were purified from thesupernatants by Protein A affinity chromatography (GE-Healthcare cat no89928) with an ÄKTA Purifier instrument (GE Healthcare). Purification ofthe anti-CD3×anti-HER2 scFv control (COVA446, SEQ ID NO: 391) wasachieved by immobilized metal ion affinity chromatography via a HIStrapExcel column (GE Healthcare). Concentrations were determined byabsorbance measurement at 280 nm. Yields are listed in Table 17.

Results

Antibody Fynomer® fusion proteins of the invention and the controlantibodies could be expressed and purified in a single step. A purityof >95% could be demonstrated by SDS-PAGE analysis as shown in FIGS. 46and 47. Table 17 summarizes the expression yield after transienttransfection into CHO cells and protein expression for 6 days at 37° C.

TABLE 17 Protein SEQ ID NO: Yield (mg/l) Anti-CD3 381, 382 46 antibody(COVA419) COVA420 387, 388 59 COVA422 389, 390 21 Anti-CD3 × 391 17 HER2scFv control (COVA446)

After purification size exclusion chromatography has been performedusing an ÄKTA FPLC system and a Superdex G200, 30/100 GL column (GEHealthcare) or an Agilent HPLC 12/60 system and a Bio SEC5, 5 mm; 300 Å;4.6×300 mm column. The bispecific proteins of the invention eluted as asingle monomeric peak demonstrating that the antibody Fynomer® fusionproteins of the present invention have very favourable biophysicalproperties (FIG. 48).

Example 6.1.2 FACS Experiment with Anti-HER2 Fynomer® Anti-CD3 AntibodyFusion Proteins of the Current Invention

The proteins COVA420 (SEQ ID NO: 386 and 387), COVA422 (SEQ ID NO: 388and 390) and COVA446 (SEQ ID NO: 391) were incubated at a concentrationof 100 nM in a total volume of 100 μl containing either (i) 1×10⁵ JurkatE6-1 cells (CD3 positive cells), (ii) 1×10⁵ BT474 HER2 positive breastcancer cells, (iii) 1×10⁵ HER2 or CD3 negative MDA MB 468 cells inPBS/1% BSA/0.2% sodium azide. As a negative control, the same cells wereincubated with PBS/1% BSA/0.2% sodium azide only instead of proteins(PBS control).

After 60 min incubation on ice, cells were washed twice with 150 uLPBS-1% BSA buffer and bound antibodies were detected by adding 5 ug/mLgoat anti human-Alexa 488 conjugate (Invitrogen) for 40 min in the dark.For the bispecific scFv control COVA446 (SEQ ID NO: 391), carrying ahexa his tag, binding was detected by the addition of 5 ug/ml mouse antiHIS tag antibody (Fisher Scientific) and incubation at 4° C. for 40 minfollowed by an additional wash step followed by an incubation with 5ug/mL goat anti mouse Alexa 488 conjugate (Invitrogen) again for 40 minat 4° C.

Finally cells were washed three times, resuspended in 100 μl PBS-1% BSA,and stained cells were then analyzed on a Guava easyCyte™ flow cytometer(Millipore).

Results

Binding properties of Fynomer®-antibody fusion proteins were evaluatedvia flow cytometry. (COVA420 (SEQ ID NO: 387 and 388), COVA422 (SEQ IDNO: 389 and 390) and the bispecific scFv control (COVA446, SEQ ID NO:391) specifically bound to HER2 expressing BT474 cells (panel A) and CD3expressing Jurkat E6 cells (panel B) as shown in FIG. 49. Nonon-specific binding was observed on cell lines that did not expresseither CD3 or HER2 (panel C).

Example 6.1.3 Redirected T-Cell Mediated Cell Cytotoxicity AnalysisTowards Cells Expressing HER2

Polypeptides COVA420 (SEQ ID NO: 387 and 388), and the bispecific scFvcontrol (SEQ ID NO: 381) were tested in a redirected T-cell mediatedcell cytotoxicity assay using a protocol adapted from Jäger et al (2009)Cancer Research, 69 (10):4270-6.

In brief, human PBMCs were used as effector cells. On the day before theexperiment PBMCs were isolated from fresh buffy coat preparationsobtained from Blutspende Zurich by Ficoll Plaque plus (GE Healthcare)and density gradient centrifugation using standard procedures. IsolatedPBMCs were then incubated over night at a cell concentration of 4×10⁶cells/ml in 10% FCS, RPMI and 37° C., 5% CO₂. The isolated PBMCs couldthen serve as effector cells in redirected cell cytotoxicity assays.

Also on the night before the experiment an appropriate amount of targetcells were detached by Accutase treatment and target cells were seededin 96 well plates at cell densities ranging from 3000-5000 cells perwell in 100 uL complete cell culture medium supplemented with 10% FCS.Assay plates were incubated over night at 37° C. and 5% CO2. Targettumor cells used were SKBR-3 (ATCC cat no HTB-30™) and MDA-MB-468 (ATCCcat no: HTB-132™).

Prior to the cell kill experiment an appropriate amount of PBMCs wascentrifuged and resuspended in 10′)/0 FCS, RPMI. Depending on the numberof target cells used the cell concentration was adjusted ranging from1.5 to 2.5×10⁶ cells/mL and cells stored at room temperature untilfurther use.

Redirected T-cell mediated cell cytotoxicity was additionally monitoredusing enriched CD8+ T-cells as effector cells and SKOV-3 (ATCC cat noHTB-77™) as target tumor cells. For these assays, isolated PBMCs werefurther purified to obtain enriched CD8+ T-cells. CD8+ T cells wereisolated using the Dynbead® Untouched™ Human CD8 T-cell kit (LifeTechnologies cat no: 11348D) according to manufacturers recommendation.After purification cells were then resuspended in 10% FCS, RPMI at aconcentration ranging from 6×10⁵ to 1×10⁷ per ml.

On the day of the experiment effector molecules were diluted in 10% FCS,RPMI to a maximum concentration of 150 nM and a dilution series of 1/10dilutions was prepared.

Finally, consumed medium was removed from the assay plates andsubstituted with 50 ul of fresh medium per well. Then appropriateamounts of effector molecules and effector cells were added. The finaleffector cell to target cell ratio was 25/1 for PBMC assays and 10/1 forassays in which CD8+ T-cells served as effector cells. The final maximumconcentration of effector molecule was 50 nM. The final assay volume was150 uL per well.

The assay plates were incubated between 24 h and 72 h at 37° C., 5% CO2.Then, cell culture supernatants were removed and stored at −80° C. forsubsequent assays (granzyme release assay, see Example 6.1.4 below).Prior to the addition of developing solution each well was washed withPBS twice. Cell viability was evaluated using XTT-reagent (Sigma cat no:X4626) according to manufactures recommendation. A 100% lysis controlwas included by treating the target cells with 1% Triton-X100 (Sigma),and the value for spontaneous lysis was obtained by treating the targetcells with effector cells only (“spont lysis”). Absorbance at 450 nm wasmeasured between 2.5 and 4 h after addition of XTT substrate. Allmeasurements were done in triplicates.

Percent cell viability was calculated using the following formula:% viability=(value−100% lysis)/(spont lysis−100% lysis)*100%

% cell viability was then plotted against the effector moleculeconcentration and data were evaluated using Prism 5 (GraphPad Software)by fitting a sigmoidal dose-response.

Results

It could be demonstrated that a dose dependent cytotoxicity of COVA420(SEQ ID NO: 387 and 388) on HER2 expressing SKBR-3 cells could beobserved (FIG. 50). In this representative assay, COVA420 (SEQ ID NO:387 and 388) had an EC₅₀ value of 87 pM. Importantly, the cytotoxiceffects were antigen dependent since no cytotoxicity was observed onHER2 negative MDA-MB-468 as shown in FIG. 50.

Under the same assay conditions an EC₅₀ value of 60 pM was obtained forthe bispecific anti-HER2×anti-CD3 scFv-scFv control molecule (SEQ ID NO:391). It was surprisingly found that bivalent and full length IgG basedFynomer-antibody fusion proteins show potencies in a redirected killassay that are in the same range as currently used scFv-scFv proteins,but which do not suffer from the drawbacks of suboptimal biophysicalproperties and short in vivo half-life.

Table 18 summarizes the EC₅₀ values obtained for the proteins tested:

TABLE 18 EC₅₀ (pM) Protein SEQ ID NO. PBMCs COVA420 387, 388 87scFv-scFv 391 60 control (COVA446)

In order to demonstrate that the Fynomer-antibody fusion proteins of theinvention exert the killing activity through the engagement of T-cells(and not through ADCC mediated activity), redirected cell killexperiments using CD8+ enriched T-cells that cannot mediate cell killingthrough ADCC were performed.

FIG. 51 shows that cell killing could be confirmed using CD8+ enrichedT-cells as effector cells. Here, COVA420 (SEQ ID NO: 387 and 388) showedan EC₅₀ value of 8 pM and the EC₅₀ value of COVA422 (SEQ ID NO: 389 and390) was 175 pM These results confirm the potent (sub-nM) killingactivity of COVA420 (SEQ ID NO: 387 and 388) and COVA22 (SEQ ID NO: 389and 390).

Example 6.1.4 Analysis of Granzyme B Release in the Presence and Absenceof Target Cells

The release of Granzyme B into the cell culture medium upon incubationof COVA420 (SEQ ID NO: 387 and 388) and the anti-CD3 antibody (SEQ IDNO: 381 and 382) as a control in the presence and absence of antigenpositive target cells and CD8+ enriched T-cells was evaluated. Releaseof Granzyme B is a main indicator of T-cell mediated cytotoxic activityinduced by BiTE® like agents as described by Haas A. et. al.Immunobiology, 2009, 214 (6): 441-53.

Samples were incubated as described above for evaluating the cytotoxicactivity of Fynomer®-antibody fusion proteins. At the end of theincubation period the cell culture supernatants were collected and theconcentration of Granzyme B was evaluated by using a Granzyme B ELISAkit (R&D Systems) according to manufacturers recommendation.

Results:

FIG. 52 depicts the expression level of Granzyme B after 3 days ofincubation of COVA420 (SEQ ID NO: 387 and 388) in the presence of CD8⁺T-Cells and in the presence and absence of antigen positive target cellsat the indicated concentrations. No unspecific release of Granzyme Bcould be detected if T-cells were only incubated with 50 nM COVA420 (SEQID NOs: 387 and 388) in the absence of target cells. A pronouncedincrease in Granzyme B expression was observed when target cells COVA420(SEQ ID No: 387 and 388) and T-cells were present. No substantialGranzyme B expression could be detected when COVA420 (SEQ ID No: 387 and388) was used at concentrations in which no cytotoxic effect wasdetectable which indicates a correlation between cytotoxic activity andGranzyme B release (0.5 pM). The control anti-CD3 antibody (SEQ ID NO:381 and 382) (50 nM) did not trigger Granzyme B release in the presenceof tumor target and effector cells, as expected.

Example 6.1.5 Pharmacokinetic Analysis

The pharmacokinetic profile of COVA420 (SEQ ID NO: 387 and 388) inC57BL/6 mice (Charles River) was investigated. Five C57BL/6 mice wereinjected i.v. with 200 μg COVA420 (SEQ ID NO: 387 and 388). After 10min, 6, 24, 48, 96, 120, 144 and 168 hours, blood was collected intoEDTA coated microvettes (Sarstedt), centrifuged for 10 min at 9300 g andthe serum levels of COVA420 (SEQ ID NO: 387 and 388) was determined byELISA. Black maxisorp microtiter plates (Nunc) were coated with 50 nMHER2 ECD (Bender MedSystems). After blocking with 2% BSA (Sigma) in PBS,40 μl of PBS and 10 μl of serum at appropriate dilution were applied.After incubation for 1 hr, wells were washed with PBS, and bound COVA420(SEQ ID NO: 387 and 388) was detected with anti-hIgG-HRP (JacksonImmunoResearch). The assay was developed with QuantaRed fluorogenicsubstrate (Pierce) and the fluorescence intensity was measured after 5min at 544 nm (excitation) and 590 nm (emission). The serum levels ofCOVA420 (SEQ ID NO: 387 and 388) were determined using a standard curveof COVA420 (SEQ ID NO: 387 and 388) (diluted to 333-0.5 ng/ml). From theconcentrations determined in serum at different time points and theresulting slope k of the elimination phase (plotted in asemi-logarithmic scale), the half-life was calculated using to theformula t_(1/2)=ln 2/−k.

Results:

FIG. 53 shows the serum concentrations of COVA420 (SEQ ID NO: 387 and388) after an iv bolus injection in mice. The half-life value forCOVA420 (SEQ ID NO: 387 and 388) as determined from the eliminationphase (beta phase, time-points 24 h-168 h) was 140 hours. This findingdemonstrates that COVA420 (SEQ ID NO: 387 and 388) has IgG-like in vivoPK properties, as the half-life was comparable to the half-livesobtained for other human antibodies in mice (eg. adalimumab: 102-193hours, Humira® Drug Approval Package (Drug Approval Package, Humira®,FDA Application No.: 125057s0110, Pharmacology Review, Jan. 18, 2008).

Example 6.1.6 In Vivo Efficacy of COVA420 in HER2-Overexpressing SKOV-3Tumor Bearing Mice Reconstituted with Human T-Cells

The anti-tumor activity of COVA420 was investigated in irradiatedNOD.CB17 Prkdc mice bearing a HER2-expressing human SKOV-3 tumorxenograft.

Methods:

3×10⁶ SKOV-3 (ATCC cat no HTB-77™) cells were injected subcutaneously(s.c.) into 2 Gy-irradiated NOD.CB17 Prkdc mice (Charles River). Whentumors reached an average size of ca. 50 mm³, animals were treated witha single intravenous (i.v.) bolus injection of anti-asialo GM1 rabbitantibody (WAKO, Germany) one day before human T-cell injection. In vitroactivated and expanded (22 days) human T-cells (Miltenyi Biotech,Germany) isolated from a buffy coat of a single healthy donor, wereinjected (1.6×10⁷ per mouse) into the peritoneal cavity. Three daysafter T-cell injection, mice were randomized and received 0.5 mg/kgCOVA420 (SEQ ID NO: 387 and 388; n=8), vehicle (PBS; n=7) treatmentstwice per week (days 6, 9, 13, 15) or daily equimolar doses (=0.16mg/kg) of COVA446 (SEQ ID NO: 391; n=8) by i.v. bolus injection into thelateral tail vein for a total of 15 days. Treatment efficacy wasassessed by tumor growth inhibition. Tumor size was measured by externalcaliper measurements and volume calculated using the standardhemi-ellipsoid formula: volume=(width)²×length×0.5. Relative tumorvolumes (RTV) to day 6 (initiation of therapy) are presented asmean±SEM.

Statistical analysis was performed using GraphPad Prism 6 software,version 6a. Statistical significance of anti-tumor efficacy wascalculated by using an unpaired, nonparametric t-test (Mann-Whitney).Anti-tumor efficacy of COVA420 vs vehicle treatment on day 16 wasfurther evaluated as tumor volume inhibition relative to the vehiclecontrol, expressed as treatment-to-control ratio (T/C): T/C (%)=RTV (day16)/RTV (day 6) (Wu (2010), Journal of Biopharmaceutical Statistics,20:5, 954-964).

Results:

COVA420 significantly reduces SKOV-3 tumor growth by actively recruitingT-cells to the tumor (FIG. 54). COVA420 treatment resulted in asignificant growth inhibition as compared to the vehicle control on day16, after 4 doses of 0.5 mg/kg COVA420 (P=0.0059). COVA420 treatment wasalso significantly more efficacious as compared to the daily injected0.16 mg/kg COVA446 control (P=0.04). T/C ratio on the same day equals to55% growth inhibition of COVA420 and 79% of COVA446 as compared to thevehicle treatment. The results demonstrate that COVA420 ispharmacologically active and exerts its anti-tumor activity byefficiently recruiting human T-cells to the tumor resulting in inhibitedtumor growth as compared to the vehicle-treated control.

Example 6.1.7 Analysis of Redirected T-Cell Mediated Cell CytotoxicitySelectively Towards Tumor Cells Expressing High Levels of HER2

Polypeptides COVA420 (SEQ ID NOs: 387 and 388) and the bispecific scFvcontrol COVA446 (SEQ ID NO: 391) were tested in a redirected T-cellmediated cell cytotoxicity assay using a protocol adapted from Jäger etal (2009) Cancer Research, 69 (10):4270-6.

In brief, human PBMCs were isolated from fresh buffy coat the day beforeand CD8+ T-cells were isolated on the day of the experiment as describedin example 6.1.3.

On the night before the experiment an appropriate number of target cellswere detached by Accutase treatment and target cells were seeded in 96well plates at a cell density of 5000 cells per well in 150 μlappropriate growth medium supplemented with 10% FCS. Assay plates wereincubated over night at 37° C. and 5% CO₂. Target tumor cells used wereSKOV-3 (ATCC® HTB-77™) expressing high level of HER2 (approx. 1.7×10⁶HER2 molecules/cell) and MCF-7 (ATCC® HTB-22™) expressing low level ofHER2 (approx. 1×10⁴ HER2 molecules/cell). HER2 surface expression wasquantified using Qifikit (Dako K0078) according to the manufacturersrecommendation. Briefly, cells were stained with a saturatingconcentration of anti-HER2 antibody (R&D MAB1129) or isotype matchedcontrol IgG, followed by anti-mouse-FITC secondary antibody, and flowcytometric analysis. At the same time, beads coated with differentwell-defined quantities of mouse monoclonal antibody molecules werestained with the secondary antibody, analysed and a standard curve wasplotted as a reference for molecules/cell.

On the day of the experiment, effector molecules were diluted in 10%FCS, RPMI to a maximum concentration of 150 nM and a dilution series of1/10 dilutions was prepared.

An appropriate amount of T-cells was centrifuged and resuspended in 10%FCS, RPMI. The cell concentration was adjusted to, 1×10⁶ cells/mL.

Finally, consumed medium was removed from the assay plates andsubstituted with 50 μl of fresh 10% FCS, RPMI per well. Then 50 μl ofeffector molecules and 50 μl of effector T-cells were added. The finaleffector cell to target cell ratio was 10:1. The final maximumconcentration of effector molecule was 50 nM. The final assay volume was150 μl per well.

The assay plates were incubated for 60 h at 37° C., 5% CO₂. Then, cellculture supernatants were removed and each well was washed once withPBS. Cell viability was evaluated using XTT-reagent (Sigma X4626)according to the manufacturer's recommendation. A 100% lysis control wasincluded by treating the target cells with 1% Triton-X100 (Sigma), andthe value for spontaneous lysis was obtained by incubating the targetcells with effector cells only (“spont lysis”). Absorbance at 450 nm wasmeasured 2 h after addition of XTT substrate. All measurements were donein triplicates.

Percent cell viability was calculated using the following formula:% viability=(value−100% lysis)/(spont lysis−100% lysis)*100%

% cell viability was then plotted against the effector moleculeconcentration and data were evaluated using Prism 5 (GraphPad Software)by fitting a sigmoidal dose-response.

Results

Dose dependent redirected T-cell cytotoxicity of COVA420 towards SKOV-3target cells expressing high level of HER2 could be observed (FIG. 55).Table 19 summarizes the EC₅₀ values obtained for the proteins tested.

Furthermore, dose dependent redirected T-cell cytotoxicity of thebispecific anti-CD3×anti-HER2 scFv control molecule COVA446 towardsMCF-7 target cells expressing low level of HER2 could be observed. Inthis representative assay, COVA446 had an EC₅₀ value of 53 pM.Surprisingly, COVA420 did not exhibit significant redirected T-cellcytotoxicity towards MCF-7 target cells expressing low level of HER2(Table 3).

TABLE 19 SEQ ID SKOV-3 MCF-7 Factor Protein NO. EC₅₀ (pM) EC₅₀ (pM)difference COVA420 387, 388 11.6 n.d. >4310 anti- CD3 × 391 2.3 53 23EGFR scFv control (COVA446)

Example 6.2 Redirected T-Cell Mediated Cell Cytotoxicity AnalysisTowards EGFR Positive Tumor Cells Example 6.2.1 Expression andPurification of Anti-CD3 Antibody Anti-EGFR Fynomer Fusion Proteins

DNA encoding the polypeptides shown in SEQ ID NOs: 392 and 410 to 420were cloned into the bacterial expression vector pQE12 so that theresulting constructs carried a C-terminal myc-hexahistidine tag asdescribed in Grabulovski et al. (Grabulovski et al. (2007) JBC, 282, p.3196-3204). The polypeptides were expressed in the cytosol of E. colibacteria in 200 μl scale cultures. Cleared lysate containing thepolypeptides was diluted 5:1 in PBS/1% FCS/0.2% sodium azide buffercontaining 10 μg/ml anti-myc mouse antibody 9E10 (Roche) and added to1×10⁵ MDA-MB-468 cells (ATCC® HTB-132™). After 60 min incubation on ice,cells were washed, and bound sequences were detected by anti-mouseIgG-Alexa488 conjugate (Invitrogen). The stained cells were thenanalyzed in a FACS analyzer. The DNA sequence of the specific binderswas verified by DNA sequencing. The amino acid sequences of Fyn SH3derived EGFR binders is presented in SEQ ID NOs: 392 and 410 to 420 asappended in the sequence listing.

The EGFR binding Fynomer ER7L2D6 (SEQ ID NO: 392) has been geneticallyfused to the N-terminus of the heavy chain (SEQ ID NO: 394) of a CD3binding antibody (COVA489, SEQ ID NOs: 394 and 395) via a 15 amino acidlinker (SEQ ID NO: 386) yielding the bispecific antibody Fynomer fusionprotein COVA493 (SEQ ID NOs: 393 and 394). In addition, ER7L2D6 (SEQ IDNO: 381) was fused to the N-terminus of the antibody light chainresulting in the bispecific antibody Fynomer fusion protein COVA494 (SEQOD NOs: 394 and 421).

The EGFR binding Fynomer ER9L3D7 (SEQ ID NO: 410) has been geneticallyfused to the N-terminus of the antibody heavy chain (SEQ ID NO: 394) ofan anti-CD3 binding antibody (COVA489, SEQ ID NOs: 394 and 395) via a 15amino acid linker (SEQ ID NO: 386) yielding the bispecific antibodyFynomer fusion protein COVA497 (SEQ ID NOs: 422 and 395). In addition,ER9L3D7 (SEQ ID NO: 381) was fused to the C-terminus of the antibodylight chain (SEQ ID NOs: 395) resulting in the bispecific antibodyFynomer fusion protein COVA499 (SEQ OD NOs: 394 and 423).

The anti-CD3 antibody COVA489, the bispecific proteins COVA493, COVA494,COVA497, COVA499 and the anti-CD3×anti-EGFR scFv control COVA445 (SEQ IDNO: 396) (carrying a hexa-his tag), were transiently transfected intoFreeStyle CHO-S cells and expressed in serum-free/animal component-freemedia for 6 days. The anti-CD3 antibody and the bispecific proteins ofthe invention were purified from the supernatants by Protein A affinitychromatography (GE-Healthcare cat no 89928) with an ÄKTA Purifierinstrument (GE Healthcare). Purification of the anti-CD3×anti-EGFR scFvcontrol (COVA445, SEQ ID NO: 396) was achieved by immobilized metal ionaffinity chromatography via a HIStrap Excel column (GE Healthcare).Concentrations were determined by absorbance measurement at 280 nm.Yields are listed in Table 20.

After purification, analytical size exclusion chromatography wasperformed using a silica-based SEC-5 column (Agilent; 5 mm; 300 Å) on anAgilent HPLC 12/60 system.

Results

Antibody Fynomer fusion proteins of the invention and the controlantibodies could be expressed and purified in a single step. Table 1summarizes the purification yield after transient transfection into CHOcells and protein expression for 6 days at 37° C.

TABLE 20 Expression yields. Protein SEQ ID NO: Yield (mg/l) Anti-CD3394, 395 66 antibody (COVA489) COVA493 393, 395 40 COVA494 394, 421 39COVA497 422, 395 35 COVA499 394, 423 74 Anti-CD3 × 396 34 EGFR scFvcontrol (COVA445)

The bispecific proteins of the invention eluted as a single monomericpeak from the size exclusion column, demonstrating that the antibodyFynomer fusion proteins of the present invention have very favorablebiophysical properties (FIG. 56). The bispecific constructs of theinvention expressed at least as good as the unmodified anti-CD3 antibodyand did not aggregate after purification as shown by SEC analysis.

Example 6.2.2 FACS Assay Experiment with Anti-EGFR Fynomer® Anti-CD3Antibody Fusion Proteins of the Current Invention

COVA489, COVA493, COVA494, COVA497, COVA499 and COVA445 were incubatedeither (i) at 30 nM concentration in a total volume of 100 μl with 1×10⁵Jurkat E6-1 cells (CD3 positive cells; ATCC® TIB-152™), or (ii) at 100nM concentration in a total volume of 100 μl with 1×10⁵EGFR-overexpressing MDA-MB-468 breast cancer cells (ATCC® HTB-132™) inPBS/1% BSA/0.2% sodium azide. As a negative control, the same cells wereincubated with PBS/1% BSA/0.2% sodium azide only instead of proteins(PBS control).

After 45 min incubation on ice, cells were washed twice with 150 μLPBS-1% BSA buffer and bound antibodies were detected by adding 4 ug/mLgoat anti-human-Alexa 488 conjugate (Invitrogen) for 40 min at 4° C. Forthe bispecific scFv control COVA445 carrying a hexa his tag, binding wasdetected by concomitant incubation of COVA445 with a mouse anti HIS tagantibody (Fisher Scientific) at a molar ratio of 1:4 (anti-HIS tagantibody:COVA445), followed by additional wash steps, followed byincubation with 4 ug/mL goat anti-mouse-Alexa 488 conjugate (Invitrogen)for 40 min at 4° C.

Finally cells were washed three times, resuspended in 100 μl PBS-1% BSA,and stained cells were then analyzed on a Guava easyCyte™ flow cytometer(Millipore).

Results

All constructs bound to Jurkat E6-1 cells which express human CD3, andall Fynomer-antibody fusions as well as the anti-CD3×anti-EGFR scFvcontrol COVA445 bound to MDA-MB-468 (FIG. 57). No specific binding wasobserved for the anti-CD3 antibody COVA489 on MDA-MB-468, as expected.

Example 6.2.3 Analysis of Redirected T-Cell Mediated Cell CytotoxicitySelectively Towards Tumor Cells Expressing High Levels of EGFR

Polypeptides COVA493, COVA494, COVA497, COVA499, the bispecific scFvcontrol COVA445 and the anti-CD3 antibody (COVA489, SEQ ID Nos: 394 and395) were tested in a redirected T-cell mediated cell cytotoxicity assayusing a protocol adapted from Jäger et al (2009) Cancer Research, 69(10):4270-6.

In brief, on the night before the experiment an appropriate number oftarget cells were detached by Accutase treatment and target cells wereseeded in 96 well plates at a cell density of 3000-5000 cells per wellin 150 μl appropriate growth medium supplemented with 10% FCS. Assayplates were incubated over night at 37° C. and 5% CO₂. Target tumorcells used were MDA-MB-468 (ATCC® HTB-132™) expressing high level ofEGFR (approx. 1.5×10⁶ EGFR molecules/cell) and HT-29 (ATCC® HTB-38™)expressing low level of EGFR (approx. 5×10⁴ EGFR molecules/cell). EGFRsurface expression was quantified as described in Example 6.1.7 butusing a saturating concentration of anti-EGFR antibody (MilliporeMABF120).

On the day of the experiment, effector molecules were diluted in 10%FCS, RPMI to a maximum concentration of 15 nM and a dilution series of1/20 dilutions was prepared.

Human CD8+ enriched T-cells were used as effector cells. CD8+ enrichedT-cells were isolated from fresh buffy coat preparations obtained fromBlutspende Bern using the MACSxpress human CD8+ isolation kit (Miltenyi130-098-194) together with the MACSxpress Separator (Milteny130-098-309) and Red blood cell lysis solution (Milteny 130-094-183) asrecommended by the manufacturer.

An appropriate amount of T-cells was centrifuged and resuspended in 10%FCS, RPMI. The cell concentration was adjusted to, 1×10⁶ cells/mL.

Finally, consumed medium was removed from the assay plates andsubstituted with 50 μl of fresh 10% FCS, RPMI per well. Then 50 μl ofeffector molecules and 50 μl of effector T-cells were added. The finaleffector cell to target cell ratio was 10:1. The final maximumconcentration of effector molecule was 5 nM. The final assay volume was150 μl per well.

In order to demonstrate that the Fynomer-antibody fusion proteins of theinvention exert the killing activity through the engagement of T-cells,target cells were incubated with COVA493, COVA494, COVA497, COVA499 andCOVA445 at a concentration of 5 nM also in the absence of T-cells.

The assay plates were incubated for 64 h at 37° C., 5% CO₂. Then, cellculture supernatants were removed and each well was washed once withPBS. Cell viability was evaluated using XTT-reagent (Sigma X4626)according to the manufacturer's recommendation. A 100% lysis control wasincluded by treating the target cells with 1% Triton-X100 (Sigma), andthe value for spontaneous lysis was obtained by incubating the targetcells with effector cells only (“spont lysis”). Absorbance at 450 nm wasmeasured 5 h after addition of XTT substrate. All measurements were donein triplicates.

Percent cell viability was calculated using the following formula:% viability=(value−100% lysis)/(spont lysis−100% lysis)*100%

% cell viability was then plotted against the effector moleculeconcentration and data were evaluated using Prism 5 (GraphPad Software)by fitting a sigmoidal dose-response.

Results

Dose dependent redirected T-cell cytotoxicity of COVA493, COVA494,COVA497 and COVA499 towards MDA-MB-468 target cells expressing highlevel of EGFR could be observed (FIG. 58. Table 21 summarizes the EC₅₀values obtained for the proteins tested.

Furthermore, dose dependent redirected T-cell cytotoxicity of COVA493and the bispecific anti-CD3×anti-EGFR scFv control molecule towardsHT-29 target cells expressing low level of EGFR could be observed. Inthis representative assay, COVA493 had an EC₅₀ value of 117.3 pM, andCOVA445 had an EC₅₀ value of 2.5 pM. Surprisingly, COVA494, COVA497 andCOVA499 did not exhibit significant redirected T-cell cytotoxicitytowards HT-29 target cells expressing low level of EGFR even at thehighest concentration of 5 nM (Table 5).

The cytotoxic effects were dependent on the presence of the anti-EGFRFynomer since no cytotoxicity was observed with the anti-CD3 antibody(COVA489, SEQ ID NOs: 384 and 385, measured at the highest concentrationof 5 nM).

TABLE 21 EC50 values for T-cell mediated cytotoxicity. SEQ ID MDA-MB-468HT-29 Factor Protein NO. EC₅₀ (pM) EC₅₀ (pM) difference COVA493 393, 3950.2 117.3 580 COVA494 394, 421 1.4 n.a. >3500 COVA497 422, 395 2.8n.a. >1750 COVA499 394, 423 6 n.a. >830 anti- CD3 × 396 0.2  2.5 12.5EGFR scFv control (COVA445) (n.a. not applicable)

The cytotoxic effects were dependent on the presence of effector T-cells(FIG. 59), since no cytotoxicity was observed when target cells wereincubated with COVA493, COVA494, COVA497, COVA499 and the bispecificanti-CD3×anti-EGFR scFv control molecule (COVA445).

Example 6.3 Redirected T-Cell Mediated Cell Cytotoxicity AnalysisTowards CD33 Positive Tumor Cells Example 6.3.1 Expression andPurification of Anti-CD3 Antibody Anti-CD33 Fynomer Fusion Proteins

DNA encoding the amino acids shown in SEQ ID NOs: 397 and 400 to 409were cloned into the bacterial expression vector pQE12 so that theresulting constructs carried a C-terminal myc-hexahistidine tag asdescribed in Grabulovski et al. (Grabulovski et al. (2007) JBC, 282, p.3196-3204). The polypeptides were expressed in the cytosol of E. colibacteria in 200 μl scale cultures. Monoclonal cleared lysates were usedfor ELISA: human recombinant CD33 (purchased from R&D as fusion to humanFcγ1) was immobilized on MaxiSorp wells (Nunc), and after blocking with4% milk (Rapilait, Migros, Switzerland) in PBS, 12.5 μl of 10% milk inPBS containing 50 μg/ml mouse anti-myc mouse antibody 9E10 (Roche) and50 μl of cleared lysate were applied. After incubation for 1 hr, unboundFynomers were washed off, and bound Fynomers were detected withanti-mouse IgG-HRP antibody conjugate (Sigma). The detection ofperoxidase activity was done by adding BM blue POD substrate (Roche) andthe reaction was stopped by adding 1 M H₂SO₄. The ELISA positive cloneswere tested by an identical ELISA for the absence of cross reactivity tohuman IgG (Sigma) and to uncoated MaxiSorp wells. Furthermore, DNAencoding the polypeptides shown in SEQ ID NOs: 397 and 400 to 409 werepurified from the bacterial lysates using immobilized metal affinitychromatography columns as described in Grabulovski et al. (Grabulovskiet al. (2007) JBC, 282, p. 3196-3204). 93 nM purified Fynomer wereincubated with 1×10⁵ U937 cells (ATCC CRL-1593.2™) in 100 μl PBS/1%FCS/0.2% sodium azide containing 23 nM mouse anti-myc antibody 9E10.After 60 min incubation on ice, cells were washed, and bound sequenceswere detected by anti-mouse IgG-Alexa488 conjugate (Invitrogen). Thestained cells were then analyzed in a FACS analyzer. The DNA sequence ofthe specific binders was verified by DNA sequencing. The amino acidsequences of Fyn SH3 derived CD33 binders are presented in SEQ ID NOs:397 and 400 to 409 as appended in the sequence listing.

The CD33 binding Fynomer EE1L1B3 (Seq ID NO: 397) has been geneticallyfused to the C-terminus of the light chain (SEQ ID NO: 382) of ananti-CD3 binding antibody (SEQ ID NOs: 381 and 382) via a 15 amino acidlinker (SEQ ID NO: 386) yielding the bispecific antibody Fynomerantibody fusion protein COVA467 (SEQ ID NOs: 381 and 175) of the presentinvention. COVA467 and the anti-CD3×anti-CD33 scFv control COVA463 (SEQID NO: 399) described in patent application WO 2010/037835 wereexpressed and purified as described in Example 6.1.1. COVA467 expressedas good as the unmodified anti-CD3 antibody (yields: 56 mg/l for COVA467vs. 46 mg/l for the parental anti-CD3 antibody COVA419) and did notaggregate after purification as shown by SEC analysis FIG. 60 A.

Example 6.3.2 Analysis of Redirected T-Cell Mediated Cell CytotoxicityTowards Tumor Cells Expressing CD33

Polypeptides COVA467 (SEQ ID NOs: 381 and 398), and the bispecific scFvcontrol COVA463 (SEQ ID NO: 399) were tested in a redirected T-cellmediated cell cytotoxicity assay using a protocol adapted from Jäger etal (2009) Cancer Research, 69 (10):4270-6.

In brief, U937 (ATCC cat no: CRL-1593.2™) target cells were seeded inround bottom 96 well plates at a cell density of 10000 cells per well in50 μL RPMI medium supplemented with 10% FCS. Effector molecules werediluted in 10% FCS, RPMI to a maximum concentration of 15 nM and adilution series of 1/10 dilutions was prepared. Human CD8+ enrichedT-cells were used as effector cells. CD8+ enriched T-cells were isolatedas described in Example 6.1.3 and adjusted to a concentration of to2×10⁶ cells/mL. Then 50 μl of effector molecules at the indicatedconcentrations and 50 μl of effector T-cells were added. The finaleffector cell to target cell ratio was 10:1. The final assay volume was150 μl per well.

The assay plates were incubated for 24 h at 37° C., 5% CO₂. Cell lysiswas evaluated using CytoTox-Fluor™ cytotoxicity assay (Promega G9260)according to manufactures recommendation. A 100% lysis control wasincluded by treating the target cells with 2% Saponin (Sigma) at 0 hincubation. Spontaneous lysis was measured by incubating the targetcells with effector T-cells only (“spont lysis”).

After 24 h incubation, assay plates were spun down at 400×g for 10 minand 100 μl of supernatant was transferred into black 96 well plates.CytoTox-Fluor™ reagent and assay buffer was thawed at room temperatureand 60 μl of reagent were diluted in 30 ml assay buffer. Subsequently 50μl of this mixture was added to each well of assay plate supernatant.Plates were incubated for 2 h at 37° C. and fluorescence was recorded at485 nm excitation/520 nm emission. All measurements were done intriplicates.

Percent cell viability was calculated using the following formula:% cell lysis=(value−spont lysis)/(100% lysis−spont lysis)*100%

% cell lysis was then plotted against the effector moleculeconcentration and data were evaluated using Prism 5 (GraphPad Software)by fitting a sigmoidal dose-response.

Results

It could be demonstrated that a dose dependent cytotoxicity of COVA467(SEQ ID NOs: 381 and 398) on CD33 expressing U937 cells could beobserved (FIG. 60 B). In this representative assay, COVA467 (SEQ ID NOs:381 and 398) had an EC₅₀ value of 2.1 pM. Importantly, the cytotoxiceffects were dependent on the presence of the anti-CD33 Fynomer since nocytotoxicity was observed with the anti-CD3 antibody (COVA419, SEQ IDNOs: 381 and 382).

Under the same assay conditions an EC₅₀ value of 1.6 pM was obtained forthe bispecific anti-CD33×anti-CD3 scFv-scFv control molecule (SEQ ID NO:399). It was surprisingly found that bivalent and full length IgG basedFynomer-antibody fusion proteins show potencies in a redirected killassay that are in the same range as currently used scFv-scFv proteins,but which do not suffer from the drawbacks of suboptimal biophysicalproperties and short in vivo half-life.

Table 22 summarizes the EC₅₀ values obtained for the proteins tested:

TABLE 22 SEQ ID Fynomer NO. EC₅₀ (pM) COVA467 381, 398 2.1 scFv-scFv 3991.6 control (COVA463)

Example 7 Anti-BACE2 Serum Albumin Fyn SH3 Derivatives Example 7.1BACE2-Specific Fynomers

Methods:

BACE2 is a membrane-bound aspartic protease (UniProt Q95YZ0).BACE2-specific Fynomers were obtained and characterized as described inBanner et al (Banner et al (2013) Acta Cryst D69, pp. 1124-1137).Briefly, starting from a phage library with two randomized loops (RT andSrc) and different loop lengths, three rounds of panning and phageamplification were performed using streptavidin-immobilized biotinylatedBACE2 (biotinylation procedure is a methodology well known in the art).Phage clones were screened by phage ELISA and their loop sequences wereanalyzed. Clones were selected and used as templates for one round ofaffinity maturation with specifically designed sub-libraries. TheFynomer sequences were cloned into bacterial expression vector (pQE12)with a C-terminal 6×His tag. The Fynomers were expressed, small-scalepurified and screened by ELISA and Biacore as described in Banner et al(2013) Acta Cryst D69, pp. 1124-1137.

Results:

Fynomers binding to BACE2 were isolated using standard phage-displaytechniques. The KD for BACE2 binding of nine Fynomers ranged from 6 to380 nM (Table 23).

TABLE 23 K_(D) values of BACE2 binding Fynomers Fynomer K_(D) [nM]1B-G10 260 2B-D2 47 1B-H10 45 1B-B11 70 1B-E11 380 2B-E9 22 2B-H11 62B-B12 9 1B-E10 200

Example 7.2 The BACE2 Binding Fynomers were Also Able to Inhibit BACE2Activity

Methods

BACE2 activity assay was performed as described in Banner et al (2013)Acta Cryst D69, pp. 1124-1137. Briefly, to determine the IC50 of theinhibiting Fynomers, a BACE2 FRET assay was performed using afluorescent substrate (WSEVNLDAEFRC-MR121) in triplicate at roomtemperature in a final volume of 50 ml in 384-well microtitre plates.All reagents were diluted in the assay buffer: 100 mM sodium acetate, 20mM EDTA, 0.05% BSA pH 4.5. The anti-BACE-2 Fynomers were seriallydiluted and 20 ml of these dilutions was mixed for 10 min with 20 mlhuman recombinant BACE-2 (final concentration 62.5 nM). After additionof 10 ml of the substrate (final concentration 300 nM), the plates wereshaken for 2 min. The enzymatic reaction was followed in a plate visionreader (PerkinElmer; excitation wavelength 630 nm; emission wavelength695 nm) for 30 min in a kinetic measurement detecting an increase ofMR121 fluorescence during the reaction time. The slope in the linearrange of the kinetics was calculated and the IC50 was determined using afour-parameter equation for curve fitting

Results:

The IC50 values of the seven inhibitory Fynomers are summarized in Table24.

TABLE 24 IC₅₀ values of BACE2 inhibitory Fynomers Fynomer IC50 [nM]1B-G10 (SEQ ID NO: 424) 1325 2B-D2 (SEQ ID NO: 425) 87 1B-H10 (SEQ IDNO: 426) 51 1B-B11 (SEQ ID NO: 427) 302 2B-E9 (SEQ ID NO: 428) 8792B-H11 (SEQ ID NO: 429) 174 2B-B12 (SEQ ID NO: 430) 35

Also described herein are the following items.

-   1. A recombinant binding protein, comprising at least one derivative    of the Src homology 3 domain (SH3) of the FYN kinase, wherein    -   (a) at least one amino acid in or positioned up to two amino        acids adjacent to the src loop and/or    -   (b) at least one amino acid in or positioned up to two amino        acids adjacent to the RT loop,    -   is substituted, deleted or added, wherein the SH3 domain        derivative has an amino acid sequence having at least 70,        preferably at least 80, more preferably at least 90 and most        preferred at least 95% sequence identity to the amino acid        sequence of SEQ ID NO: 1,    -   preferably with the proviso that the recombinant binding protein        does not comprise the amino acid sequence of SEQ ID NO: 2,    -   and preferably with the proviso that the recombinant protein is        not a natural SH3 domain containing protein existing in nature.-   2. The binding protein according to item 1, wherein said SH3 domain    derivative has at least 85, preferably at least 90, more preferably    at least 95, most preferably at least 98 to 100% identity to the Src    homology 3 domain (SH3) of the FYN kinase outside the src and RT    loops.-   3. The binding protein according to item 1 or 2, wherein    -   (a) at least one amino acid in the src loop and    -   (b) at least one amino acid in the RT loop,    -   is substituted, deleted or added.-   4. The binding protein according to any one of items 1 to 3,    comprising at least two derivatives of the SH3 domain, preferably a    bivalent binding protein.-   5. The binding protein according to any one of items 1 to 4,    comprising one or preferably two altered residues in positions 37    and/or 50 of the SH3 domain derivative, preferably two hydrophobic    altered residues, more preferably Trp37 and/or Tyr50, Trp37 and    Tyr50 being most preferred.-   6. The binding protein according to any one of items 1 to 5 having a    specific binding affinity to a target of 10⁻⁷ to 10⁻¹² M, preferably    10⁻⁸ to 10⁻¹² M, preferably a therapeutically and/or diagnostically    relevant target, more preferably an amino acid-based target    comprising a PxxP motif.-   7. The binding protein according to any one of items 1 to 6 having a    specific binding affinity of 10⁻⁷ to 10⁻¹² M, preferably 10⁻⁸ to    10⁻¹² M, to the extracellular domain of oncofetal fibronectin    (ED-B).-   8. The binding protein according to item 7 having one or more,    preferably two, altered, preferably hydrophobic, residues in    positions 37 and/or 50 of the SH3 domain derivative, more preferably    Trp37 and/or Tyr50, and most preferred Trp37 and Tyr50.-   9. The binding protein according to any one of items 1 to 8,    comprising the amino acid sequence of SEQ ID NO: 3.-   10. The binding protein according to any one of items 1 to 9,    wherein said binding protein has binding specificity for a protein    or a small organic compound.-   11. A fusion protein comprising a binding protein according to any    one of items 1 to 10 fused to a pharmaceutically and/or    diagnostically active component.-   12. The fusion protein according to item 11, wherein said component    is a cytokine, preferably a cytokine selected from the group    consisting of IL-2, IL-12, TNF-alpha, IFN alpha, IFN beta, IFN    gamma, IL-10, IL-15, IL-24, GM-CSF, IL-3, IL-4, IL-5, IL-6, IL-7,    IL-9, IL-11, IL-13, LIF, CD80, B70, TNF beta, LT-beta, CD-40 ligand,    Fas-ligand, TGF-beta, IL-1 alpha and IL-1 beta.-   13. The fusion protein according to item 11, wherein said component    is a toxic compound, preferably a small organic compound or a    polypeptide, preferably a toxic compound selected from the group    consisting of calicheamicin, neocarzinostatin, esperamicin,    dynemicin, kedarcidin, maduropeptin, doxorubicin, daunorubicin,    auristatin, Ricin-A chain, modeccin, truncated Pseudomonas exotoxin    A, diphtheria toxin and recombinant gelonin.-   14. The fusion protein according to item 11, wherein said component    is a chemokine, preferably a chemokine selected from the group    consisting of IL-8, GRO alpha, GRO beta, GRO gamma, ENA-78,    LDGF-PBP, GCP-2, PF4, Mig, IP-10, SDF-1alpha/beta, BUNZO/STRC33,    I-TAC, BLC/BCA-1, MIP-1alpha, MIP-1 beta, MDC, TECK, TARC, RANTES,    HCC-1, HCC-4, DC-CK1, MIP-3 alpha, MIP-3 beta, MCP-1-5, Eotaxin,    Eotaxin-2, I-309, MPIF-1, 6Ckine, CTACK, MEC, Lymphotactin and    Fractalkine.-   15. The fusion protein according to item 11, wherein said component    is a fluorescent dye, preferably a component selected from Alexa    Fluor or Cy dyes.-   16. The fusion protein according to item 11, wherein said component    is a photosensitizer, preferably bis(triethanolamine)Sn(IV)    chlorine₆ (SnChe₆).-   17. The fusion protein according to item 11, wherein said component    is a pro-coagulant factor, preferably tissue factor.-   18. The fusion protein according to item 11, wherein said component    is an enzyme for pro-drug activation, preferably an enzyme selected    from the group consisting of carboxy-peptidases, glucuronidases and    glucosidases.-   19. The fusion protein according to item 11, wherein said component    is a radionuclide either from the group of gamma-emitting isotopes,    preferably ^(99m)Tc, ¹²³I, ¹¹¹In, or from the group of positron    emitters, preferably ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ¹²⁴I, or from the group    of beta-emitter, preferably ¹³¹I, ⁹⁰Y, ¹⁷⁷Lu, ⁶⁷Cu, or from the    group of alpha-emitter, preferably ²¹³Bi, ²¹¹At.-   20. The fusion protein according to item 11, wherein said component    is a functional Fc domain, preferably a human functional Fc domain.-   21. The fusion protein according to any one of items 11 to 20,    further comprising a component modulating serum half-life,    preferably a component selected from the group consisting of    polyethylene glycol (PEG), immunoglobulin and albumin-binding    peptides.-   22. The fusion protein according to any one of items 11 to 21,    comprising the binding protein according to item 7, 8 or 9.-   23. A polynucleotide coding for a binding protein or fusion protein    according to any one of items 1 to 22.-   24. A vector comprising a polynucleotide according to item 23.-   25. A host cell comprising a polynucleotide according to item 23    and/or a vector according to item 24.-   26. Use of a binding or fusion protein according to any one of items    1 to 14, 16 to 18 and 20 to 22 for preparing a medicament.-   27. Use of a binding protein according to item 7, 8 or 9 and/or a    fusion protein according to item 22 for preparing a medicament for    the treatment of cancer.-   28. Use of a binding or fusion protein according to any one of items    1 to 10, 15, 19, 21 and 22 for preparing a diagnostic means.-   29. Use of a binding protein according to item 7, 8 or 9 and/or a    fusion protein according to item 22 for preparing a diagnostic means    for the diagnosis of cancer.-   30. A pharmaceutical composition comprising a binding or fusion    protein according to any one of items 1 to 14, 16 to 18 and 20 to 22    and optionally a pharmaceutically acceptable excipient.-   31. A diagnostic composition comprising a binding or fusion protein    according to any one of items 1 to 11, 15, 19, 21 and 22 and    optionally a pharmaceutically acceptable excipient.

The invention claimed is:
 1. A method for the production of a librarycomprising recombinant derivatives of the SH3 domain of the Fyn kinaseof SEQ ID NO: 1, said derivatives having a specific binding affinity toa protein or peptide, wherein said protein or peptide is not a naturalSH3 binding ligand, wherein said method comprises the steps of (a)generating recombinant derivatives of the SH3 domain of the Fyn kinaseof SEQ ID NO: 1 by substituting at least one amino acid in the RT loopof SEQ ID NO: 1, and substituting at least one amino acid in the srcloop of SEQ ID NO: 1, and (b) constructing a library comprising therecombinant derivatives of the SH3 domain generated in step (a); whereinthe recombinant derivatives of the SH3 domain of the Fyn kinase of SEQID NO: 1 retain at least 80% sequence identity to the amino acidsequence of SEQ ID NO: 1, and retain at least 90% identity to the aminoacid sequence of SEQ ID NO: 1 outside the src and RT loops; and whereinthe src loop is located at amino acid positions 31 to 34 of SEQ ID NO:1, and the RT loop is located at amino acid positions 12 to 17 of SEQ IDNO:
 1. 2. The method of claim 1, wherein in step (a) the at least oneamino acid in the RT loop of SEQ ID NO: 1 and the at least one aminoacid in the src loop of SEQ ID NO: 1 are simultaneously substituted. 3.The method of claim 1, wherein step (b) further comprises displaying therecombinant derivatives of the SH3 domain of the Fyn kinase of SEQ IDNO: 1 on phages.
 4. The method of claim 1, wherein the library comprisesmore than one (1) billion different recombinant derivatives of the SH3domain of the Fyn kinase of SEQ ID NO: 1.